Engineered tyrosine ammonia lyase

ABSTRACT

The present invention provides engineered tyrosine ammonia-lyase (TAL) polypeptides and compositions thereof. In some embodiments, the engineered TAL polypeptides have been optimized to provide enhanced catalytic activity while reducing sensitivity to proteolysis and increasing tolerance to acidic pH levels. The invention also provides methods for utilization of the compositions comprising the engineered TAL polypeptides for therapeutic and industrial purposes.

The present application is a Divisional of co-pending U.S. patentapplication Ser. No. 16/705,656, filed Dec. 6, 2019, which claimspriority to U.S. Prov. Appln. Ser. No. 62/779,647, filed Dec. 14, 2018,both of which are hereby incorporated by reference in their entiretiesfor all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX

The Sequence Listing written in file CX7-182US2D1_ST26.xml, created onSep. 12, 2022, size of 5.27 MB, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention provides engineered tyrosine ammonia-lyase (TAL)polypeptides and compositions thereof. In some embodiments, theengineered TAL polypeptides have been optimized to provide enhancedcatalytic activity while reducing sensitivity to proteolysis andincreasing tolerance to acidic pH levels. The invention also providesmethods for utilization of the compositions comprising the engineeredTAL polypeptides for therapeutic and industrial purposes.

BACKGROUND OF THE INVENTION

Tyrosine ammonia lyase (TAL; also referred to as tyrase, L-tyrosineammonia lyase, and “L-tyrosine ammonia lyase [trans-p-hyroxycinnamateforming]”), along with histidine ammonia lyase (HAL) and phenylalanineammonia-lyase (PAL) are members of the aromatic amino acid lyase family(EC 4.3.1.23-1.25 and 4.3.1.3). The enzymes having TAL activity arecurrently classified in EC4.3.1.23 (previously classified as EC4.3.1.5). TAL catalyzes the formation of p-coumaric acid fromL-tyrosine.

Tyrosinemia (also referred to as “hereditary tyrosinemia,” and“hypertyrosinemia”) is a genetic disorder characterized by elevatedblood levels of tyrosine, due to the deficiency of an enzyme requiredfor the catabolism of tyrosine in the liver. If untreated, tyrosine andother metabolites accumulate in the tissues and organs of affectedindividuals, resulting in serious medical issues. Tyrosinemia is aninborn error of metabolism inherited in an autosomal recessive pattern.There are three types of tyrosinemia, each caused by the deficiency of adifferent enzyme. Currently used treatment methods depend upon the typeof tyrosinemia involved. A low protein diet is often used.

Type I tyrosinemia (also referred to as “FAH deficiency,” “fumarylacetoacetase deficiency,” “fumaryl aceotacetate hydrolase deficiency,”“hereditary infantile tyrosinemia,” and “hepatorenal tyrosinemia”) iscaused by a deficiency of fumarylacetoacetate hydrolase, due tomutations in the fah gene. This is the most severe form of the disease,with symptoms usually appearing in the first few months of life,commonly including failure to thrive, diarrhea, bloody stools, vomiting,jaundice, enlarged liver, the tendency to easily bruise, lethargy,irritability, fever, and other symptoms, such as a distinctivecabbage-like odor of the skin and urine. Some affected infants haverepeated neurologic episodes of acute polyneuropathy, characterized bysevere leg pain, as well as altered mental status, abdominal pain, andrespiratory failure. Infants with the acute form are typically affectedat birth and there is a rapid onset of symptoms that can lead todevelopmental delays, enlarged spleen, ascites, kidney disease, andblood clotting abnormalities. Untreated, it can lead to hepatic andrenal failure, nervous system problems, and an increased risk of livercancer (e.g., hepatocellular carcinoma). In some cases, hypertension andhypertrophic cardiomyopathy are present. If untreated, this disease canbe fatal. In the less-common chronic form, the symptoms exhibit a moregradual onset and tend to be less severe. Affected infants initiallyexhibit vomiting, diarrhea, enlarged liver and spleen, and failure tothrive. Eventually, progressive liver cirrhosis occurs, leading tochronic liver failure, developmental delays, and renal Fanconi syndrome(a rare kidney disorder characterized by weakening and softening of thebones [rickets], vomiting, dehydration, weakness, and fever). In somecases, the most effective treatment has been full or partial livertransplant. Worldwide, this form affects approximately 1 in 100,000human births (Genetics Home Reference, U.S. National Library ofMedicine).

Type II tyrosinemia (also referred to as “keratosispalmoplantaris-corneal dystrophy,” oculocutaneous tyrosinemia,”“Richner-Hanhart syndrome,” “tyrosinemia due to TAT deficiency,” and“tyrosinema due to tyrosine aminotransferase deficiency,”) is caused bya deficiency of tyrosine aminotransferase, due to mutations in the tatgene. It affects the eyes, skin, and mental development. As with Type Ityrosinemia, symptoms usually begin in early life, and include excessivetearing, photophobia, eye pain and redness, and painful skin lesions onthe palms and soles. About half of affected individuals have some levelof intellectual disability. This form occurs in less than 1 in 250,000persons (Genetics Home Reference, supra).

Type III tyrosinemia (also referred to as “tyrosinemia due to4-hydroxyphenylpyruvate dioxygenase deficiency,” “tyrosinemia due to4-hydroxyphenylpyuriv acid oxidase deficiency,” and “tyrosinemia due toHPD deficiency”) is a rare disorder, caused by a deficiency of4-hydroxyphenylpyruvate dioxygenase, due to mutations in the hpd gene.Symptoms of this form include intellectual disability, seizures, andintermittent ataxia. This form is very rare, only a few cases have beenreported (Genetics Home Reference, supra).

There are additional cases in which there are temporary elevatedtyrosine levels, due to non-genetic factors such as vitamin C deficiencyor premature birth, which results in immature liver enzymes.Differential diagnoses are used to differentiate these transient casesfrom tyrosinema I, II, or III.

In addition to tyrosinemia, there are other diseases associated withinsufficient or absent tyrosine metabolism. For example, alkaptonuriaalso referred to as alcaptonuria, is a disease caused by deficiency ofhomogentisate 1,2-dioxygenase, which is an enzyme involved in tyrosinedegradation. This enzyme is encoded by the HGD gene. Insufficientactivity of this enzyme results in the accumulation of homogentisicacid. Excess homogentisic acid and related compounds are deposited inconnective tissues, causing the cartilage and skin to darken. Over time,arthritis may result due to the accumulation of homogentisic acid andrelated metabolites in the joints of affected individuals. Homogentisicacid is also excreted in urine, making the urine turn black.Alkaptonuria is a rare disease that affects 1 in 250,000 to 1,000,000people worldwide (See, Genetics Home Reference, supra).

Treatment of these diseases has largely been the life-long use of aphenylalanine-, and tyrosine-restricted diet. Treatment with nitisinone(NTBC; 2-(2-nitro-4-trifluoromethylbenzol)-1,3-cyclohexane dione;Orfadin®) has been reported to be helpful for type I tyrosinemia andalkaptonuria, due to its inhibition of the 4-hydroxyphenylpyruvateoxidase pathway. However, NTBC must be used in combination with achallenging and costly phenylalanine-, and tyrosine-restricted diet toprevent both liver failure and carcinogenesis. There remains a need inthe art for easy to administer, effective treatment(s) to ameliorate thesymptoms of these diseases and allow patients to utilize normal diets.

SUMMARY OF THE INVENTION

The present invention provides engineered tyrosine ammonia-lyase (TAL)polypeptides and compositions thereof. In some embodiments, theengineered TAL polypeptides have been optimized to provide enhancedcatalytic activity while reducing sensitivity to proteolysis andincreasing tolerance to acidic pH levels. The invention also providesmethods for utilization of the compositions comprising the engineeredTAL polypeptides for therapeutic and industrial purposes.

In some embodiments, the present invention provides engineered TALpolypeptides (also referred to herein as “recombinant TAL polypeptides”)and biologically active fragments and analogs thereof having improvedproperties when compared to a wild-type TAL enzyme and/or a referenceTAL polypeptide under essentially the same conditions. The invention isfurther directed to methods of using the engineered TAL polypeptides andbiologically active fragments and analogs thereof in therapeutic and/orindustrial compositions.

The present invention provides recombinant tyrosine ammonia lyasesand/or biologically active recombinant tyrosine ammonia lyase fragmentscomprising an amino acid sequence comprising at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to SEQ ID NO: 2. In someembodiments, the tyrosine ammonia lyase comprises at least onesubstitution at position or set of positions selected from 79/95/107,79/107, 79/107/410, 79/410, 95/107/184, 95/107/184/534, 95/184/534,99/107/184, 107/184/534, and 107/401/534, wherein the positions arenumbered with reference to SEQ ID NO: 2. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets at one or more positions selected from 79T/95A/107S,79T/107S, 79T/107S/410K, 79T/410K, 95A/107A/184S, 95A/107S/184S/534S,95A/184S/534S, 99H/107A/184S, 107A/184S/534S, and 107S/401P/534S,wherein the positions are numbered with reference to SEQ ID NO: 2. Insome embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets at one or more positions selected fromS79T/K95A/V107S, S79T/V107S, S79T/V107S/P410K, S79T/P410K,K95A/V107A/Y184S, K95A/V107S/Y184S/A534S, K95A/Y184S/A534S,N99H/V107A/Y184S, V107A/Y184S/A534S, and V107S/A401P/A534S, wherein thepositions are numbered with reference to SEQ ID NO: 2.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 28, 29, 33, 33/34/37/46/279/554, 33/34/37/90/554,33/34/46/66/90/289, 33/34/90/289/554, 33/34/279/289, 33/66, 33/66/289,33/66/289/554, 34/37/46/289, 34/37/66/279/289, 34/46, 34/56/90,34/56/90/289, 34/66/289, 34/90/289/554, 34/90/519,36/70/234/302/461/500, 36/79, 36/79/304/407, 36/79/407/461/500/531,36/111/304/461/531, 36/407/461/500/531, 37, 46, 46/66/289/519/554, 49,56, 66, 66/90/279/289/554, 66/90/289/554, 69, 70/79/234/361/407,70/111/461/500/531, 70/304/407/461, 72, 79/111/234/361/500,79/111/234/531, 79/111/361, 79/111/531, 79/304/361, 79/304/461/531,79/407/461, 88, 90/289/519, 90/289/554, 131, 142, 198, 201, 272, 278,279/289/554, 289/519/554, 289/554, 295, 304/361, 305, 350, 361/500/531,461/500, 490, 491, 499, 510, 518, 519, 521, 522, 523, 524, 531, 540,541, 544, 547, 548, 554, 557, 558, and 560, wherein the positions arenumbered with reference to SEQ ID NO: 14. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets selected from 28A, 28C, 28G, 28N, 28T, 29A, 29E, 29G,29R, 29W, 33D/34E/37S/46R/279S/554Q, 33D/34E/37S/90S/554Q,33D/34E/46R/66N/90S/289R, 33D/34E/90S/289R/554Q, 33D/34E/279S/289R,33D/66N, 33D/66N/289R, 33D/66N/289R/554Q, 33N, 34E/37R/46R/289R,34E/37S/66N/279S/289R, 34E/46R, 34E/56G/90S, 34E/56G/90S/289R,34E/66N/289R, 34E/90S/289R/554Q, 34E/90S/519P,36V/70K/234H/302M/461M/500T, 36V/79T, 36V/79T/304G/407V,36V/79T/407V/461M/500T/531D, 36V/111K/304G/461M/531D,36V/407V/461M/500T/531D, 37A, 37D, 46R, 46R/66N/289R/519P/554Q, 46T,49D, 49N, 56G, 56R, 66N/90S/279D/289R/554Q, 66N/90S/289R/554Q, 66R, 66S,69S, 70K/79T/234H/361M/407V, 70K/111K/461M/500T/531D,70K/304G/407V/461M, 72T, 79T/111K/234H/361M/500T, 79T/111K/234H/531D,79T/111K/361M, 79T/111K/531D, 79T/304G/361M, 79T/304G/461M/531D,79T/407V/461M, 88E, 88Q, 90S/289R/519P, 90S/289R/554Q, 131G, 131M, 131S,131W, 142R, 198R, 201R, 272S, 278S, 279S/289R/554Q, 289R/519P/554Q,289R/554Q, 295Q, 304G/361M, 305H, 350G, 361M/500Q/531D, 461M/500T, 490A,490I, 490S, 490V, 491L, 491V, 499T, 510K, 518E, 518V, 519D, 519E, 519L,519P, 519Q, 521D, 522G, 522K, 522R, 522V, 522Y, 523A, 523D, 523H, 523I,523S, 524A, 524I, 524L, 524Q, 524T, 524Y, 531D, 540E, 540R, 541A, 544E,544G, 544R, 544S, 547A, 547C, 547G, 547K, 547Q, 547R, 547S, 548A, 548G,548K, 548Q, 548R, 548T, 554E, 554R, 554S, 554Y, 557E, 557R, 558A, 558D,558H, 558S, 560G, 560M, 560R, and 560T, wherein the positions arenumbered with reference to SEQ ID NO: 14. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets selected from K28A, K28C, K28G, K28N, K28T, P29A,P29E, P29G, P29R, P29W, K33D/K34E/Q37S/L46R/Q279S/K554Q,K33D/K34E/Q37S/T90S/K554Q, K33D/K34E/L46R/Q66N/T90S/A289R,K33D/K34E/T90S/A289R/K554Q, K33D/K34E/Q279S/A289R, K33D/Q66N,K33D/Q66N/A289R, K33D/Q66N/A289R/K554Q, K33N, K34E/Q37R/L46R/A289R,K34E/Q37S/Q66N/Q279S/A289R, K34E/L46R, K34E/K56G/T90S,K34E/K56G/T90S/A289R, K34E/Q66N/A289R, K34E/T90S/A289R/K554Q,K34E/T90S/A519P, A36V/S70K/Y234H/R302M/L461M/C500T, A36V/S79T,A36V/S79T/E304G/S407V, A36V/S79T/S407V/L461M/C500T/N531D,A36V/Q111K/E304G/L461M/N531D, A36V/S407V/L461M/C500T/N531D, Q37A, Q37D,L46R, L46R/Q66N/A289R/A519P/K554Q, L46T, T49D, T49N, K56G, K56R,Q66N/T90S/Q279D/A289R/K554Q, Q66N/T90S/A289R/K554Q, Q66R, Q66S, A69S,S70K/S79T/Y234H/L361M/S407V, S70K/Q111K/L461M/C500T/N531D,S70K/E304G/S407V/L461M, E72T, S79T/Q111K/Y234H/L361M/C500T,S79T/Q111K/Y234H/N531D, S79T/Q111K/L361M, S79T/Q111K/N531D,S79T/E304G/L361M, S79T/E304G/L461M/N531D, S79T/S407V/L461M, V88E, V88Q,T90S/A289R/A519P, T90S/A289R/K554Q, H131G, H131M, H131S, H131W, Q142R,E198R, K201R, P272S, A278S, Q279S/A289R/K554Q, A289R/A519P/K554Q,A289R/K554Q, L295Q, E304G/L361M, Q305H, A350G, L361M/C500Q/N531D,L461M/C500T, K490A, K490I, K490S, K490V, I491L, I491V, Q499T, L510K,K518E, K518V, A519D, A519E, A519L, A519P, A519Q, S521D, E522G, E522K,E522R, E522V, E522Y, E523A, E523D, E523H, E523I, E523S, R524A, R524I,R524L, R524Q, R524T, R524Y, N531D, A540E, A540R, L541A, A544E, A544G,A544R, A544S, T547A, T547C, T547G, T547K, T547Q, T547R, T547S, S548A,S548G, S548K, S548Q, S548R, S548T, K554E, K554R, K554S, K554Y, N557E,N557R, Q558A, Q558D, Q558H, Q558S, V560G, V560M, V560R, and V560T,wherein the positions are numbered with reference to SEQ ID NO: 14.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 28/34/49/70/234/289/544/554, 33/34/49/234/531/547/554,33/34/66/70/519/531, 33/34/66/289/522/531, 33/34/70/234/289/519,33/34/70/234/518/519/547/554, 33/34/70/289/522/554, 33/34/289/522/531,34/49/66/531, 34/49/234/531/554, 34/49/289/544/547,34/66/70/234/518/519/547, 34/66/531/554, 34/66/554, 34/70/234/289/522,34/70/234/554, 34/70/289/522, 34/234/522/554, 34/289, 34/289/522/531,34/289/522/544/554, 34/522, 34/544, 66/70/234, and 234/531/544/547,wherein the positions are numbered with reference to SEQ ID NO: 86. Insome embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from28/34/49/70/234/289/544/554, 33/34/49/234/531/547/554,33/34/66/70/519/531, 33/34/66/289/522/531, 33/34/70/234/289/519,33/34/70/234/518/519/547/554, 33/34/70/289/522/554, 33/34/289/522/531,34/49/66/531, 34/49/234/531/554, 34/49/289/544/547,34/66/70/234/518/519/547, 34/66/531/554, 34/66/554, 34/70/234/289/522,34/70/234/554, 34/70/289/522, 34/234/522/554, 34/289, 34/289/522/531,34/289/522/544/554, 34/522, 34/544, 66/70/234, and 234/531/544/547,wherein the positions are numbered with reference to SEQ ID NO: 86. Insome embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from28N/34E/49N/70S/234Y/289R/544E/554Q, 33D/34E/66N/70S/519P/531D,33D/34E/66N/289R/522K/531D, 33D/34E/70S/234Y/289R/519P,33D/34E/70S/234Y/518V/519P/547Q/554Q, 33D/34E/70S/289R/522K/554Q,33D/34E/289R/522K/531D, 33N/34E/49N/234Y/531D/547Q/554Q,34E/49N/66N/531D, 34E/49N/234Y/531D/554Q, 34E/49N/289R/544E/547Q,34E/66N/705/234Y/518V/519P/547Q, 34E/66N/531D/554Q, 34E/66N/554Q,34E/70S/234Y/289R/522K, 34E/70S/234Y/554Q, 34E/70S/289R/522K,34E/234Y/522K/554Q, 34E/289R, 34E/289R/522K/531D,34E/289R/522K/544E/554Q, 34E/522K, 34E/544E, 66N/70S/234Y, and234Y/531D/544E/547Q, wherein the positions are numbered with referenceto SEQ ID NO: 86.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 31, 37/49/56/66/111, 37/49/56/234/557, 37/49/56/500/524,37/49/66/111/234, 37/49/111/524, 37/111, 37/111/361/500/557/558, 37/234,43/59/93/506, 43/306, 43/306/506, 49/56/66/111/153/234/500/524, 49/66,56/111/234, 56/234/500, 61, 65, 65/336, 66, 66/111/234/500, 93/506, 95,103, 103/107/417/421, 103/306/506, 103/421, 104/105, 107, 107/417/421,111/234/548/558, 111/361, 145, 146, 155, 172, 197, 211, 234, 234/524,237/238/506, 286, 304, 306, 306/506, 328, 336, 361/524/557/558, 407,417/421, 421, 471, 504, 506, 549, and 558, wherein the positions arenumbered with reference to SEQ ID NO: 334. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets selected from 31S, 37D/49N/56G/66N/111K,37D/49N/56G/234Y/557E, 37D/49N/56G/500T/524Q, 37D/49N/66N/111K/234Y,37D/49N/111K/524Q, 37D/111K, 37D/111K/361L/500T/557E/558D, 37D/234Y,43T/59A/93Q/506R, 43T/306L, 43T/306L/506R,49N/56G/66N/111K/153N/234Y/500T/524Q, 49N/66N, 56G/111K/234Y,56G/234Y/500T, 61V, 65M, 65N/336V, 66N, 66N/111K/234Y/500T, 93Q/506R,95K, 95V, 103L, 103W, 103W/107T/417A/421S, 103W/306L/506R, 103W/421S,104F/105M, 107T, 107T/417A/421A, 111K/234Y/548K/558D, 111K/361L, 145V,146K, 155L, 155S, 172S, 197T, 211K, 234Y, 234Y/524Q, 237R/238V/506R,286Q, 286R, 304N, 306L, 306L/506R, 328A, 336V, 361L/524Q/557E/558D,407K, 407T, 417A/421S, 421S, 471Y, 504R, 506R, 506V, 549D, and 558D,wherein the positions are numbered with reference to SEQ ID NO: 334. Insome embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from T31S,Q37D/T49N/K56G/Q66N/Q111K, Q37D/T49N/K56G/H234Y/N557E,Q37D/T49N/K56G/C500T/R524Q, Q37D/T49N/Q66N/Q111K/H234Y,Q37D/T49N/Q111K/R524Q, Q37D/Q111K, Q37D/Q111K/M361L/C500T/N557E/Q558D,Q37D/H234Y, A43T/S59A/L93Q/L506R, A43T/P306L, A43T/P306L/L506R,T49N/K56G/Q66N/Q111K/T153N/H234Y/C500T/R524Q, T49N/Q66N,K56G/Q111K/H234Y, K56G/H234Y/C500T, C61V, A65M, A65N/I336V, Q66N,Q66N/Q111K/H234Y/C500T, L93Q/L506R, A95K, A95V, R103L, R103W,R103W/A107T/G417A/T421S, R103W/P306L/L506R, R103W/T421S, Y104F/H105M,A107T, A107T/G417A/T421A, Q111K/H234Y/S548K/Q558D, Q111K/M361L, E145V,T146K, H155L, H155S, A172S, P197T, L211K, H234Y, H234Y/R524Q,Q237R/N238V/L506R, A286Q, A286R, E304N, P306L, P306L/L506R, S328A,I336V, M361L/R524Q/N557E/Q558D, V407K, V407T, G417A/T421S, T421S, Q471Y,A504R, L506R, L506V, G549D, and Q558D, wherein the positions arenumbered with reference to SEQ ID NO: 334.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 31/61/95/286/328, 31/61/95/407/504, 31/61/95/471/504,31/61/407/504, 31/95/155/286/504/549, 31/95/237/504/547/549,31/95/286/504, 31/95/286/504/549, 31/95/328/504, 31/95/504, 31/95/549,37/49/56/59/145, 37/103/111/145/421/500/524, 49/56/59/234/500,49/59/66/111/145/234, 49/59/145/234/421, 56/59/66/234, 56/59/111/145,56/59/111/145/234/421, 56/66/145/500, 56/103/111/145/234/500,59/103/145/234/421/500, 59/103/234/421, 59/145, 59/234, 61,61/95/155/286/407/471, 61/95/155/407, 61/95/286, 61/95/286/328/504,61/95/328/407, 61/95/328/549, 61/95/407, 61/95/504/547, 61/286/407,61/328, 61/328/504, 61/328/504/549, 61/407, 61/504, 66/103/145/234/421,95/155/286/328/407/504, 95/155/286/549, 95/286, 95/286/328/407/504,95/286/328/504, 95/286/547, 95/287/504/549, 95/328/504, 95/407,95/407/504, 95/504/547, 95/549, 103/111/145/234/421, 111/145/234,111/234, 145/234/500, 286/328/407/504/549, and 549, wherein thepositions are numbered with reference to SEQ ID NO: 388. In someembodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from 31S/61V/95K/286Q/328A,31S/61V/95K/407T/504R, 31S/61V/95K/471Y/504R, 31S/61V/407T/504R,31S/95K/155S/286Q/504R/549D, 31S/95K/237K/504R/547Q/549D,31S/95K/286Q/504R, 31S/95K/286Q/504R/549D, 31S/95K/328A/504R,31S/95K/504R, 31S/95K/549D, 37D/49N/56G/59A/145V,37D/103L/111K/145V/421S/500T/524Q, 49N/56G/59A/234Y/500T,49N/59A/66N/111K/145V/234Y, 49N/59A/145V/234Y/421S, 56G/59A/66N/234Y,56G/59A/111K/145V, 56G/59A/111K/145V/234Y/421S, 56G/66N/145V/500T,56G/103L/111K/145V/234Y/500T, 59A/103L/145V/234Y/421S/500T,59A/103L/234Y/421S, 59A/145V, 59A/234Y, 61V,61V/95K/155S/286Q/407T/471Y, 61V/95K/155S/407T, 61V/95K/286Q,61V/95K/286Q/328A/504R, 61V/95K/328A/407T, 61V/95K/328A/549D,61V/95K/407T, 61V/95K/504R/547Q, 61V/286Q/407T, 61V/328A, 61V/328A/504R,61V/328A/504R/549D, 61V/407T, 61V/504R, 66N/103L/145V/234Y/421S,95K/155S/286Q/328A/407T/504R, 95K/155S/286Q/549D, 95K/286Q,95K/286Q/328A/407T/504R, 95K/286Q/328A/504R, 95K/286Q/547Q,95K/287S/504R/549D, 95K/328A/504R, 95K/407T, 95K/407T/504R,95K/504R/547Q, 95K/549D, 103L/111K/145V/234Y/421S, 111K/145V/234Y,111K/234Y, 145V/234Y/500T, 286Q/328A/407T/504R/549D, and 549D, whereinthe positions are numbered with reference to SEQ ID NO: 388. In someembodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected fromT31S/C61V/A95K/A286Q/S328A, T31S/C61V/A95K/V407T/A504R,T31S/C61V/A95K/Q471Y/A504R, T31S/C61V/V407T/A504R,T31S/A95K/H155S/A286Q/A504R/G549D, T31S/A95K/Q237K/A504R/T547Q/G549D,T31S/A95K/A286Q/A504R, T31S/A95K/A286Q/A504R/G549D,T31S/A95K/S328A/A504R, T31S/A95K/A504R, T31S/A95K/G549D,Q37D/T49N/K56G/S59A/E145V, Q37D/R103L/Q111K/E145V/T421S/C500T/R524Q,T49N/K56G/S59A/H234Y/C500T, T49N/S59A/Q66N/Q111K/E145V/H234Y,T49N/S59A/E145V/H234Y/T421S, K56G/S59A/Q66N/H234Y,K56G/S59A/Q111K/E145V, K56G/S59A/Q111K/E145V/H234Y/T421S,K56G/Q66N/E145V/C500T, K56G/R103L/Q111K/E145V/H234Y/C500T,S59A/R103L/E145V/H234Y/T421S/C500T, S59A/R103L/H234Y/T421S, S59A/E145V,S59A/H234Y, C61V, C61V/A95K/H155S/A286Q/V407T/Q471Y,C61V/A95K/H155S/V407T, C61V/A95K/A286Q, C61V/A95K/A286Q/S328A/A504R,C61V/A95K/S328A/V407T, C61V/A95K/S328A/G549D, C61V/A95K/V407T,C61V/A95K/A504R/T547Q, C61V/A286Q/V407T, C61V/S328A, C61V/S328A/A504R,C61V/S328A/A504R/G549D, C61V/V407T, C61V/A504R,Q66N/R103L/E145V/H234Y/T421S, A95K/H155S/A286Q/S328A/V407T/A504R,A95K/H155S/A286Q/G549D, A95K/A286Q, A95K/A286Q/S328A/V407T/A504R,A95K/A286Q/S328A/A504R, A95K/A286Q/T547Q, A95K/G287S/A504R/G549D,A95K/S328A/A504R, A95K/V407T, A95K/V407T/A504R, A95K/A504R/T547Q,A95K/G549D, R103L/Q111K/E145V/H234Y/T421S, Q111K/E145V/H234Y,Q111K/H234Y, E145V/H234Y/C500T, A286Q/S328A/V407T/A504R/G549D, andG549D, wherein the positions are numbered with reference to SEQ ID NO:388.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 14/56/114, 14/114/283/289, 14/114/283/289/291,14/114/283/289/562, 14/114/291/518/562, 14/114/518, 14/234/283/291/305,14/283/289/562, 29/66/111, 29/66/111/540, 29/111, 29/111/523, 33/46/49,33/46/49/336/519/548/549, 33/46/49/549, 33/46/518/534/548/549,33/46/519, 33/49/336, 33/336/519, 36/37/56/59/145/234/328,36/37/59/131/531, 36/37/103/234, 36/37/131/145/234/328, 36/37/145/234,36/37/145/234/328/471/531, 36/37/234/328/531, 36/56/145/234/328,36/59/234/328, 36/59/531, 36/103/145/328/531, 36/131, 36/234/531,37/56/59/103/145/234/328/471/531, 37/56/59/234/328/524/531,37/56/59/234/531, 37/56/234/531, 37/59/234, 37/234/471, 46/49,46/49/518/519/548/549, 46/49/519, 46/155, 46/548, 49/519, 56,56/59/103/471/531, 56/59/471/531, 56/114/206/283, 56/114/291,56/114/291/518/562, 56/114/518, 56/518, 59/328/524/531, 66/540, 111,111/510/521/523/541/558, 111/523/557, 114, 114/206/283/289/562,114/283/289/291, 114/283/289/305/518, 114/283/291, 114/289, 114/289/562,114/291, 114/291/518, 114/518/562, 114/562, 131/234/328, 234, 234/328,234/328/471, 234/531, 289/562, 305, 336/519, 518/519/548/549, 519, 521,523, 531, 541, 548/549, and 549, wherein the positions are numbered withreference to SEQ ID NO: 604. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom 14S/56R/114P, 14S/114P/283D/289Q, 14S/114P/283D/289Q/291V,14S/114P/283S/289Q/562S, 14S/114P/291V/518Q/562S, 14S/114P/518Q,14S/234L/283D/291V/305E, 14S/283D/289Q/562S, 29E/66N/111K/540R,29E/111K, 29E/111K/523D, 29G/66N/111K, 33N/46R/49D/549D, 33N/46T/49D,33N/46T/49D/336V/519E/548K/549D, 33N/46T/49D/549D,33N/46T/518V/534S/548K/549D, 33N/46T/519E, 33N/49D/336V, 33N/336V/519P,36V/37A/56G/59A/145V/234Y/328S, 36V/37A/59A/131M/531D,36V/37A/103L/234Y, 36V/37A/131M/145V/234Y/328S, 36V/37A/145V/234Y,36V/37A/145V/234Y/328S/471Y/531D, 36V/37A/234Y/328Q/531D,36V/56G/145V/234Y/328S, 36V/59A/234Y/328S, 36V/59A/531D,36V/103L/145V/328S/531D, 36V/131M, 36V/234Y/531D,37A/56G/59A/103L/145V/234Y/328Q/471Y/531D,37A/56G/59A/234Y/328S/524L/531D, 37A/56G/59A/234Y/531D,37A/56G/234Y/531D, 37A/59A/234Y, 37A/234Y/471Y, 46R/49D,46R/49D/518V/519E/548K/549D, 46R/49D/519P, 46R/155L, 46T/49D/519E,46T/548K, 49D/519P, 56G/59A/103L/471Y/531D, 56G/59A/471Y/531D, 56R,56R/114P/206P/283D, 56R/114P/291V, 56R/114P/291V/518Q/562S,56R/114P/518Q, 56R/518Q, 59A/328Q/524L/531D, 66R/540R, 111K,111K/510K/521D/523A/541A/558D, 111K/523A/557E, 114P,114P/206P/283S/289Q/562S, 114P/283D/289Q/291V, 114P/283D/289Q/305E/518Q,114P/283D/291V, 114P/289Q, 114P/289Q/562S, 114P/291V, 114P/291V/518Q,114P/518Q/562S, 114P/562S, 131M/234Y/328Q, 234Y, 234Y/328Q,234Y/328S/471Y, 234Y/531D, 289Q/562S, 305E, 336V/519P,518V/519E/548K/549D, 519P, 521D, wherein the positions are numbered withreference to SEQ ID NO: 604. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom F14S/K56R/S114P, F14S/S114P/E283D/R289Q,F14S/S114P/E283D/R289Q/I291V, F14S/S114P/E283S/R289Q/I562S,F14S/S114P/I291V/K518Q/I562S, F14S/S114P/K518Q,F14S/H234L/E283D/I291V/Q305E, F14S/E283D/R289Q/I562S,P29E/Q66N/Q111K/A540R, P29E/Q111K, P29E/Q111K/E523D, P29G/Q66N/Q111K,K33N/L46R/T49D/G549D, K33N/L46T/T49D,K33N/L46T/T49D/I336V/A519E/S548K/G549D, K33N/L46T/T49D/G549D,K33N/L46T/K518V/A534S/S548K/G549D, K33N/L46T/A519E, K33N/T49D/I336V,K33N/I336V/A519P, A36V/Q37A/K56G/S59A/E145V/H234Y/A328S,A36V/Q37A/S59A/H131M/N531D, A36V/Q37A/R103L/H234Y,A36V/Q37A/H131M/E145V/H234Y/A328S, A36V/Q37A/E145V/H234Y,A36V/Q37A/E145V/H234Y/A328S/Q471Y/N531D, A36V/Q37A/H234Y/A328Q/N531D,A36V/K56G/E145V/H234Y/A328S, A36V/S59A/H234Y/A328S, A36V/S59A/N531D,A36V/R103L/E145V/A328S/N531D, A36V/H131M, A36V/H234Y/N531D,Q37A/K56G/S59A/R103L/E145V/H234Y/A328Q/Q471Y/N531D,Q37A/K56G/S59A/H234Y/A328S/R524L/N531D, Q37A/K56G/S59A/H234Y/N531D,Q37A/K56G/H234Y/N531D, Q37A/S59A/H234Y, Q37A/H234Y/Q471Y, L46R/T49D,L46R/T49D/K518V/A519E/S548K/G549D, L46R/T49D/A519P, L46R/H155L,L46T/T49D/A519E, L46T/S548K, T49D/A519P, K56G/S59A/R103L/Q471Y/N531D,K56G/S59A/Q471Y/N531D, K56R, K56R/S114P/E206P/E283D, K56R/S114P/I291V,K56R/S114P/I291V/K518Q/I562S, K56R/S114P/K518Q, K56R/K518Q,S59A/A328Q/R524L/N531D, Q66R/A540R, Q111K,Q111K/L510K/S521D/E523A/L541A/Q558D, Q111K/E523A/N557E, S114P,S114P/E206P/E283S/R289Q/I562S, S114P/E283D/R289Q/I291V,S114P/E283D/R289Q/Q305E/K518Q, S114P/E283D/I291V, S114P/R289Q,S114P/R289Q/I562S, S114P/I291V, S114P/I291V/K518Q, S114P/K518Q/I562S,S114P/I562S, H131M/H234Y/A328Q, H234Y, H234Y/A328Q, H234Y/A328S/Q471Y,H234Y/N531D, R289Q/I562S, Q305E, I336V/A519P, K518V/A519E/S548K/G549D,A519P, S521D, E523D, N531D, L541A, S548K/G549D, and G549D, wherein thepositions are numbered with reference to SEQ ID NO: 604.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 20/49/56/59/111/291, 20/59/234/518/549/562,49/56/59/66/291/531/549, 49/56/59/111/549, 49/56/66/111/234/291/518,49/56/66/234, 49/59/66/145/234, 49/59/66/289, 49/59/289/562,49/66/111/234/291/518/562, 49/111/531, 49/234, 56/59/111/234/289,56/59/111/234/531, 56/59/111/283/518/531, 56/59/111/291,56/59/111/291/518/531, 56/59/145/289/518/562, 56/59/234, 56/59/234/518,56/59/518, 56/111/145/234/289/531/562, 56/111/145/234/518/549/562,56/111/518/549, 56/234/291, 59, 59/111, 59/145/234/291, 59/234/291,59/234/291/387/549/562, 111/145, 111/145/234/291/562, 111/145/234/562,111/234, 111/289, 111/291, 111/518, 234, 234/518/549, 291/518/549, 518,518/531/549/562, 531, and 549, wherein the positions are numbered withreference to SEQ ID NO: 736. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom 20G/49D/56G/59A/111K/291V, 20G/59A/234Y/518Q/549D/562S,49D/56G/59A/66N/291V/531D/549D, 49D/56G/59A/111K/549D,49D/56G/66N/111K/234Y/291V/518Q, 49D/56G/66N/234Y,49D/59A/66N/145V/234Y, 49D/59A/66N/289Q, 49D/59A/289Q/562S,49D/66N/111K/234Y/291V/518Q/562S, 49D/111K/531D, 49D/234Y,56G/59A/111K/234Y/289Q, 56G/59A/111K/234Y/531D,56G/59A/111K/283D/518Q/531D, 56G/59A/111K/291V,56G/59A/111K/291V/518Q/531D, 56G/59A/145V/289Q/518Q/562S, 56G/59A/234Y,56G/59A/234Y/518Q, 56G/59A/518Q, 56G/111K/145V/234Y/289Q/531D/562S,56G/111K/145V/234Y/518Q/549D/562S, 56G/111K/518Q/549D, 56G/234Y/291V,59A, 59A/111K, 59A/145V/234Y/291V, 59A/234Y/291V,59A/234Y/291V/387T/549D/562S, 111K/145V, 111K/145V/234Y/291V/562S,111K/145V/234Y/562S, 111K/234Y, 111K/289Q, 111K/291V, 111K/518Q, 234Y,234Y/518Q/549D, 291V/518Q/549D, 518Q, 518Q/531D/549D/562S, 531D, and549D, wherein the positions are numbered with reference to SEQ ID NO:736. In some embodiments, the tyrosine ammonia lyase comprises at leastone substitution or substitution sets selected fromD20G/T49D/R56G/S59A/Q111K/I291V, D20G/S59A/H234Y/K518Q/G549D/I562S,T49D/R56G/S59A/Q66N/I291V/N531D/G549D, T49D/R56G/S59A/Q111K/G549D,T49D/R56G/Q66N/Q111K/H234Y/I291V/K518Q, T49D/R56G/Q66N/H234Y,T49D/S59A/Q66N/E145V/H234Y, T49D/S59A/Q66N/R289Q, T49D/S59A/R289Q/I562S,T49D/Q66N/Q111K/H234Y/I291V/K518Q/I562S, T49D/Q111K/N531D, T49D/H234Y,R56G/S59A/Q111K/H234Y/R289Q, R56G/S59A/Q111K/H234Y/N531D,R56G/S59A/Q111K/E283D/K518Q/N531D, R56G/S59A/Q111K/I291V,R56G/S59A/Q111K/I291V/K518Q/N531D, R56G/S59A/E145V/R289Q/K518Q/I562S,R56G/S59A/H234Y, R56G/S59A/H234Y/K518Q, R56G/S59A/K518Q,R56G/Q111K/E145V/H234Y/R289Q/N531D/I562S,R56G/Q111K/E145V/H234Y/K518Q/G549D/I562S, R56G/Q111K/K518Q/G549D,R56G/H234Y/I291V, S59A, S59A/Q111K, S59A/E145V/H234Y/I291V,S59A/H234Y/I291V, S59A/H234Y/I291V/I387T/G549D/I562S, Q111K/E145V,Q111K/E145V/H234Y/I291V/I562S, Q111K/E145V/H234Y/I562S, Q111K/H234Y,Q111K/R289Q, Q111K/I291V, Q111K/K518Q, H234Y, H234Y/K518Q/G549D,I291V/K518Q/G549D, K518Q, K518Q/N531D/G549D/I562S, N531D, and G549D,wherein the positions are numbered with reference to SEQ ID NO: 736.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 33/37, 33/37/46/305, 33/37/103, 33/37/103/305,33/37/103/305/336/541/548, 33/37/103/305/523/548, 33/37/103/305/548,33/37/103/336/518/541/548, 33/37/103/471/519/548, 33/37/103/518/523/541,33/37/103/519/523, 33/37/103/523, 33/37/103/540, 33/37/305,33/37/305/328, 33/37/305/328/548, 33/37/305/336/471/541,33/37/305/336/518/523, 33/37/305/336/521/523/548, 33/37/305/336/521/548,33/37/305/336/541, 33/37/305/471/521/548, 33/37/305/523/540,33/37/305/540, 33/37/305/541, 33/37/336, 33/37/336/540,33/37/336/540/548, 33/37/471/523/540/548, 33/37/518/521/540,33/37/518/521/548, 33/37/518/548, 33/37/521/548, 33/37/523,33/37/523/540/548, 33/37/540, 33/37/548, 33/46,33/46/66/305/521/523/540/548, 33/46/103/305/336/518/519/540,33/46/103/305/336/548, 33/46/103/305/540, 33/46/103/540/548, 33/46/305,33/46/305/336, 33/46/305/518/540/548, 33/46/336/471/519/523/548,33/46/336/521, 33/46/336/521/523, 33/46/336/540, 33/46/336/540/548,33/46/518/521/548, 33/46/521/548, 33/46/523, 33/46/541, 33/46/548,33/103/336/518/521/548, 33/471/523/541, 37, 37/103, 37/103/305/336,37/103/305/336/521/523/540/548, 37/103/305/336/523/540, 37/103/305/471,37/103/305/471/540/548, 37/103/305/521/541/548, 37/103/305/540,37/103/328/336/518/523/548, 37/103/328/521/541/548, 37/103/336,37/103/336/521/541, 37/103/519/541/548, 37/103/540, 37/103/541/548,37/305, 37/305/336, 37/305/336/521/540/548, 37/305/336/523/548,37/305/336/540/548, 37/305/336/541/548, 37/305/471,37/305/471/523/540/548, 37/305/471/540/548, 37/305/519/548,37/305/521/523/540/548, 37/305/521/540, 37/305/521/541/548,37/305/523/541/548, 37/305/541, 37/305/541/548, 37/305/548, 37/336,37/336/471/541/548, 37/336/521/523, 37/336/521/523/548, 37/336/523/548,37/336/540, 37/336/548, 37/471, 37/471/518, 37/471/518/519/523,37/471/521/540/548, 37/519, 37/521/523/548, 37/521/540, 37/523/541,37/523/548, 37/540, 37/540/548, 37/541, 37/548, 46,46/49/59/111/523/531/549, 46/49/111/234/289/436/549,46/49/111/234/289/531/549, 46/49/111/234/336, 46/49/111/234/336/523/549,46/49/111/336/549, 46/49/234/289/336, 46/49/234/336/521/523/549,46/49/234/540/549, 46/49/234/549, 46/49/289/523/531/549/562,46/49/531/549, 46/59/234, 46/59/549/562, 46/103/305,46/103/305/336/471/540/548, 46/103/305/336/523, 46/103/305/471/540/548,46/103/305/520/540, 46/103/305/548, 46/103/523/548, 46/103/540/548,46/103/541, 46/103/541/548, 46/103/548, 46/111/234/289/531/549,46/111/234/521/549, 46/111/523/531/549, 46/234/289/549,46/234/521/523/531/540/549, 46/234/549, 46/289/549, 46/305, 46/305/336,46/305/336/518, 46/305/336/522, 46/305/336/548, 46/305/471,46/305/523/548, 46/305/548, 46/336/521/523, 46/336/523/548, 46/336/540,46/336/540/548, 46/521/523, 46/521/523/540, 46/521/523/549, 46/521/548,46/523/540, 46/523/541/548, 46/541, 46/541/548, 46/548, 46/549, 49,49/59/111/234/289/521, 49/59/111/289/523, 49/59/234/289/336,49/59/234/289/336/523/531/549, 49/59/289/305/336, 49/59/289/336,49/59/336, 49/59/521/531, 49/111, 49/111/234,49/111/234/289/336/523/531/549, 49/111/234/289/521/523/531/549/562,49/111/234/289/523/549, 49/111/234/336/521/523, 49/111/234/336/523/531,49/111/234/523/531/549, 49/111/234/531, 49/111/234/549, 49/111/289,49/111/289/336/521/549, 49/111/523/531/549, 49/111/531/549, 49/234,49/234/289, 49/234/289/336/531/549, 49/234/289/523,49/234/289/523/531/540, 49/234/289/523/549, 49/234/305/549, 49/234/521,49/234/549, 49/289/305/336/523, 49/289/336, 49/289/336/521/531,49/336/521/562, 49/521/531/549/562, 49/521/549, 49/523/549, 49/549, 59,59/111/234/289/305/336/549, 59/111/234/289/305/549, 59/111/289,59/111/336, 59/549, 103, 103/336/519/548, 103/521/523/540, 111/234/289,111/234/289/523, 111/289/336/521, 111/289/336/523/549, 111/336/562,111/521/523/549, 234, 234/289, 234/289/336/523, 234/289/523/531/549/562,234/289/523/549, 234/289/531, 234/289/549, 234/336, 234/336/531/562,234/521/523, 234/521/523/549, 234/523/531, 234/531/549, 234/540,234/549, 234/562, 289, 289/336/549, 289/521/523, 289/521/523/540,289/521/523/549, 289/523, 305/336/541/548, 336/548, 519/548, 521,521/548, 521/549, 521/562, 523, 531/549, 540, and 549, wherein thepositions are numbered with reference to SEQ ID NO: 736. In someembodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from 33N/37A,33N/37A/46R/305E, 33N/37A/103L, 33N/37A/103L/305E,33N/37A/103L/305E/336V/541A/548K, 33N/37A/103L/305E/523D/548K,33N/37A/103L/305E/548K, 33N/37A/103L/336V/518V/541A/548K,33N/37A/103L/471Y/519P/548K, 33N/37A/103L/518V/523D/541A,33N/37A/103L/519P/523D, 33N/37A/103L/523D, 33N/37A/103L/540R,33N/37A/305E, 33N/37A/305E/328Q, 33N/37A/305E/328Q/548K,33N/37A/305E/336V/471Y/541A, 33N/37A/305E/336V/518V/523D,33N/37A/305E/336V/521D/523D/548K, 33N/37A/305E/336V/521D/548K,33N/37A/305E/336V/541A, 33N/37A/305E/471Y/521D/548K,33N/37A/305E/523D/540R, 33N/37A/305E/540R, 33N/37A/305E/541A,33N/37A/336V, 33N/37A/336V/540R, 33N/37A/336V/540R/548K,33N/37A/471Y/523D/540R/548K, 33N/37A/518V/521D/540R,33N/37A/518V/521D/548K, 33N/37A/518V/548K, 33N/37A/521D/548K,33N/37A/523D, 33N/37A/523D/540R/548K, 33N/37A/540R, 33N/37A/548K,33N/46R, 33N/46R/66K/305E/521D/523D/540R/548K,33N/46R/103L/305E/336V/518V/519P/540R, 33N/46R/103L/305E/336V/548K,33N/46R/103L/305E/540R, 33N/46R/103L/540R/548K, 33N/46R/305E,33N/46R/305E/336V, 33N/46R/305E/518V/540R/548K,33N/46R/336V/471Y/519P/523D/548K, 33N/46R/336V/521D,33N/46R/336V/521D/523D, 33N/46R/336V/540R, 33N/46R/336V/540R/548K,33N/46R/518V/521D/548K, 33N/46R/521D/548K, 33N/46R/523D, 33N/46R/541A,33N/46R/548K, 33N/103L/336V/518V/521D/548K, 33N/471Y/523D/541A, 37A,37A/103L, 37A/103L/305E/336V, 37A/103L/305E/336V/521D/523D/540R/548K,37A/103L/305E/336V/523D/540R, 37A/103L/305E/471Y,37A/103L/305E/471Y/540R/548K, 37A/103L/305E/521D/541A/548K,37A/103L/305E/540R, 37A/103L/328Q/336V/518V/523D/548K,37A/103L/328Q/521D/541A/548K, 37A/103L/336V, 37A/103L/336V/521D/541A,37A/103L/519P/541A/548K, 37A/103L/540R, 37A/103L/541A/548K, 37A/305E,37A/305E/336V, 37A/305E/336V/521D/540R/548K, 37A/305E/336V/523D/548K,37A/305E/336V/540R/548K, 37A/305E/336V/541A/548K, 37A/305E/471Y,37A/305E/471Y/523D/540R/548K, 37A/305E/471Y/540R/548K,37A/305E/519P/548K, 37A/305E/521D/523D/540R/548K, 37A/305E/521D/540R,37A/305E/521D/541A/548K, 37A/305E/523D/541A/548K, 37A/305E/541A,37A/305E/541A/548K, 37A/305E/548K, 37A/336V, 37A/336V/471Y/541A/548K,37A/336V/521D/523D, 37A/336V/521D/523D/548K, 37A/336V/523D/548K,37A/336V/540R, 37A/336V/548K, 37A/471Y, 37A/471Y/518V,37A/471Y/518V/519P/523D, 37A/471Y/521D/540R/548K, 37A/519P,37A/521D/523D/548K, 37A/521D/540R, 37A/523D/541A, 37A/523D/548K,37A/540R, 37A/540R/548K, 37A/541A, 37A/548K, 46R,46R/49D/59A/111K/523D/531D/549D, 46R/49D/111K/234Y/289Q/436V/549D,46R/49D/111K/234Y/289Q/531D/549D, 46R/49D/111K/234Y/336V,46R/49D/111K/234Y/336V/523D/549D, 46R/49D/111K/336V/549D,46R/49D/234Y/289Q/336V, 46R/49D/234Y/336V/521D/523D/549D,46R/49D/234Y/540R/549D, 46R/49D/234Y/549D,46R/49D/289Q/523D/531D/549D/562S, 46R/49D/531D/549D, 46R/59A/234Y,46R/59A/549D/562S, 46R/103L/305E, 46R/103L/305E/336V/471Y/540R/548K,46R/103L/305E/336V/523D, 46R/103L/305E/471Y/540R/548K,46R/103L/305E/520Q/540R, 46R/103L/305E/548K, 46R/103L/523D/548K,46R/103L/540R/548K, 46R/103L/541A, 46R/103L/541A/548K, 46R/103L/548K,46R/111K/234Y/289Q/531D/549D, 46R/111K/234Y/521D/549D,46R/111K/523D/531D/549D, 46R/234Y/289Q/549S,46R/234Y/521D/523D/531D/540R/549D, 46R/234Y/549D, 46R/289Q/549D,46R/305E, 46R/305E/336V, 46R/305E/336V/518V, 46R/305E/336V/522E,46R/305E/336V/548K, 46R/305E/471Y, 46R/305E/523D/548K, 46R/305E/548K,46R/336V/521D/523D, 46R/336V/523D/548K, 46R/336V/540R,46R/336V/540R/548K, 46R/521D/523D, 46R/521D/523D/540R,46R/521D/523D/549D, 46R/521D/548K, 46R/523D/540R, 46R/523D/541A/548K,46R/541A, 46R/541A/548K, 46R/548K, 46R/549D, 49D,49D/59A/111K/234Y/289Q/521D, 49D/59A/111K/289Q/523D,49D/59A/234Y/289Q/336V, 49D/59A/234Y/289Q/336V/523D/531D/549D,49D/59A/289Q/305E/336V, 49D/59A/289Q/336V, 49D/59A/336V,49D/59A/521D/531D, 49D/111K, 49D/111K/234Y,49D/111K/234Y/289Q/336V/523D/531D/549D,49D/111K/234Y/289Q/521D/523D/531D/549D/562S,49D/111K/234Y/289Q/523D/549D, 49D/111K/234Y/336V/521D/523D,49D/111K/234Y/336V/523D/531D, 49D/111K/234Y/523D/531D/549D,49D/111K/234Y/531D, 49D/111K/234Y/549D, 49D/111K/289Q,49D/111K/289Q/336V/521D/549D, 49D/111K/523D/531D/549D,49D/111K/531D/549D, 49D/234Y, 49D/234Y/289Q,49D/234Y/289Q/336V/531D/549D, 49D/234Y/289Q/523D,49D/234Y/289Q/523D/531D/540R, 49D/234Y/289Q/523D/549D,49D/234Y/305E/549D, 49D/234Y/521D, 49D/234Y/549D,49D/289Q/305E/336V/523D, 49D/289Q/336V, 49D/289Q/336V/521D/531D,49D/336V/521D/562S, 49D/521D/531D/549D/562S, 49D/521D/549D,49D/523D/549D, 49D/549D, 59A, 59A/111K/234Y/289Q/305E/336V/549D,59A/111K/234Y/289Q/305E/549D, 59A/111K/289Q, 59A/111K/336V, 59A/549D,103L, 103L/336V/519P/548K, 103L/521D/523D/540R, 111K/234Y/289Q,111K/234Y/289Q/523D, 111K/289Q/336V/521D, 111K/289Q/336V/523D/549D,111K/336V/562S, 111K/521D/523D/549D, 234Y, 234Y/289Q,234Y/289Q/336V/523D, 234Y/289Q/523D/531D/549D/562S, 234Y/289Q/523D/549D,234Y/289Q/531D, 234Y/289Q/549D, 234Y/336V, 234Y/336V/531D/562S,234Y/521D/523D, 234Y/521D/523D/549D, 234Y/523D/531D, 234Y/531D/549D,234Y/540R, 234Y/549D, 234Y/562S, 289Q, 289Q/336V/549D, 289Q/521D/523D,289Q/521D/523D/540R, 289Q/521D/523D/549D, 289Q/523D,305E/336V/541A/548K, 336V/548K, 519P/548K, 521D, 521D/548K, 521D/549D,521D/562S, 523D, 531D/549D, 540R, and 549D, wherein the positions arenumbered with reference to SEQ ID NO: 736. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets selected from K33N/Q37A, K33N/Q37A/L46R/Q305E,K33N/Q37A/R103L, K33N/Q37A/R103L/Q305E,K33N/Q37A/R103L/Q305E/I336V/L541A/S548K,K33N/Q37A/R103L/Q305E/E523D/S548K, K33N/Q37A/R103L/Q305E/S548K,K33N/Q37A/R103L/I336V/K518V/L541A/S548K,K33N/Q37A/R103L/Q471Y/A519P/S548K, K33N/Q37A/R103L/K518V/E523D/L541A,K33N/Q37A/R103L/A519P/E523D, K33N/Q37A/R103L/E523D,K33N/Q37A/R103L/A540R, K33N/Q37A/Q305E, K33N/Q37A/Q305E/A328Q,K33N/Q37A/Q305E/A328Q/S548K, K33N/Q37A/Q305E/I336V/Q471Y/L541A,K33N/Q37A/Q305E/I336V/K518V/E523D,K33N/Q37A/Q305E/I336V/S521D/E523D/S548K,K33N/Q37A/Q305E/I336V/S521D/S548K, K33N/Q37A/Q305E/I336V/L541A,K33N/Q37A/Q305E/Q471Y/S521D/S548K, K33N/Q37A/Q305E/E523D/A540R,K33N/Q37A/Q305E/A540R, K33N/Q37A/Q305E/L541A, K33N/Q37A/I336V,K33N/Q37A/I336V/A540R, K33N/Q37A/I336V/A540R/S548K,K33N/Q37A/Q471Y/E523D/A540R/S548K, K33N/Q37A/K518V/S521D/A540R,K33N/Q37A/K518V/S521D/S548K, K33N/Q37A/K518V/S548K,K33N/Q37A/S521D/S548K, K33N/Q37A/E523D, K33N/Q37A/E523D/A540R/S548K,K33N/Q37A/A540R, K33N/Q37A/S548K, K33N/L46R,K33N/L46R/Q66K/Q305E/S521D/E523D/A540R/S548K,K33N/L46R/R103L/Q305E/I336V/K518V/A519P/A540R,K33N/L46R/R103L/Q305E/I336V/S548K, K33N/L46R/R103L/Q305E/A540R,K33N/L46R/R103L/A540R/S548K, K33N/L46R/Q305E, K33N/L46R/Q305E/I336V,K33N/L46R/Q305E/K518V/A540R/S548K,K33N/L46R/I336V/Q471Y/A519P/E523D/S548K, K33N/L46R/I336V/S521D,K33N/L46R/I336V/S521D/E523D, K33N/L46R/I336V/A540R,K33N/L46R/I336V/A540R/S548K, K33N/L46R/K518V/S521D/S548K,K33N/L46R/S521D/S548K, K33N/L46R/E523D, K33N/L46R/L541A,K33N/L46R/S548K, K33N/R103L/I336V/K518V/S521D/S548K,K33N/Q471Y/E523D/L541A, Q37A, Q37A/R103L, Q37A/R103L/Q305E/I336V,Q37A/R103L/Q305E/I336V/S521D/E523D/A540R/S548K,Q37A/R103L/Q305E/I336V/E523D/A540R, Q37A/R103L/Q305E/Q471Y,Q37A/R103L/Q305E/Q471Y/A540R/S548K, Q37A/R103L/Q305E/S521D/L541A/S548K,Q37A/R103L/Q305E/A540R, Q37A/R103L/A328Q/I336V/K518V/E523D/S548K,Q37A/R103L/A328Q/S521D/L541A/S548K, Q37A/R103L/I336V,Q37A/R103L/I336V/S521D/L541A, Q37A/R103L/A519P/L541A/S548K,Q37A/R103L/A540R, Q37A/R103L/L541A/S548K, Q37A/Q305E, Q37A/Q305E/I336V,Q37A/Q305E/I336V/S521D/A540R/S548K, Q37A/Q305E/I336V/E523D/S548K,Q37A/Q305E/I336V/A540R/S548K, Q37A/Q305E/I336V/L541A/S548K,Q37A/Q305E/Q471Y, Q37A/Q305E/Q471Y/E523D/A540R/S548K,Q37A/Q305E/Q471Y/A540R/S548K, Q37A/Q305E/A519P/S548K,Q37A/Q305E/S521D/E523D/A540R/S548K, Q37A/Q305E/S521D/A540R,Q37A/Q305E/S521D/L541A/S548K, Q37A/Q305E/E523D/L541A/S548K,Q37A/Q305E/L541A, Q37A/Q305E/L541A/S548K, Q37A/Q305E/S548K, Q37A/I336V,Q37A/I336V/Q471Y/L541A/S548K, Q37A/I336V/S521D/E523D,Q37A/I336V/S521D/E523D/S548K, Q37A/I336V/E523D/S548K, Q37A/I336V/A540R,Q37A/I336V/S548K, Q37A/Q471Y, Q37A/Q471Y/K518V,Q37A/Q471Y/K518V/A519P/E523D, Q37A/Q471Y/S521D/A540R/S548K, Q37A/A519P,Q37A/S521D/E523D/S548K, Q37A/S521D/A540R, Q37A/E523D/L541A,Q37A/E523D/S548K, Q37A/A540R, Q37A/A540R/S548K, Q37A/L541A, Q37A/S548K,L46R, L46R/T49D/S59A/Q111K/E523D/N531D/G549D,L46R/T49D/Q111K/H234Y/R289Q/L436V/G549D,L46R/T49D/Q111K/H234Y/R289Q/N531D/G549D, L46R/T49D/Q111K/H234Y/I336V,L46R/T49D/Q111K/H234Y/I336V/E523D/G549D, L46R/T49D/Q111K/I336V/G549D,L46R/T49D/H234Y/R289Q/I336V, L46R/T49D/H234Y/I336V/S521D/E523D/G549D,L46R/T49D/H234Y/A540R/G549D, L46R/T49D/H234Y/G549D,L46R/T49D/R289Q/E523D/N531D/G549D/I562S, L46R/T49D/N531D/G549D,L46R/S59A/H234Y, L46R/S59A/G549D/I562S, L46R/R103L/Q305E,L46R/R103L/Q305E/I336V/Q471Y/A540R/S548K, L46R/R103L/Q305E/I336V/E523D,L46R/R103L/Q305E/Q471Y/A540R/S548K, L46R/R103L/Q305E/P520Q/A540R,L46R/R103L/Q305E/S548K, L46R/R103L/E523D/S548K, L46R/R103L/A540R/S548K,L46R/R103L/L541A, L46R/R103L/L541A/S548K, L46R/R103L/S548K,L46R/Q111K/H234Y/R289Q/N531D/G549D, L46R/Q111K/H234Y/S521D/G549D,L46R/Q111K/E523D/N531D/G549D, L46R/H234Y/R289Q/G549S,L46R/H234Y/S521D/E523D/N531D/A540R/G549D, L46R/H234Y/G549D,L46R/R289Q/G549D, L46R/Q305E, L46R/Q305E/I336V, L46R/Q305E/I336V/K518V,L46R/Q305E/I336V/K522E, L46R/Q305E/I336V/S548K, L46R/Q305E/Q471Y,L46R/Q305E/E523D/S548K, L46R/Q305E/S548K, L46R/I336V/S521D/E523D,L46R/I336V/E523D/S548K, L46R/I336V/A540R, L46R/I336V/A540R/S548K,L46R/S521D/E523D, L46R/S521D/E523D/A540R, L46R/S521D/E523D/G549D,L46R/S521D/S548K, L46R/E523D/A540R, L46R/E523D/L541A/S548K, L46R/L541A,L46R/L541A/S548K, L46R/S548K, L46R/G549D, T49D,T49D/S59A/Q111K/H234Y/R289Q/S521D, T49D/S59A/Q111K/R289Q/E523D,T49D/S59A/H234Y/R289Q/I336V,T49D/S59A/H234Y/R289Q/I336V/E523D/N531D/G549D,T49D/S59A/R289Q/Q305E/I336V, T49D/S59A/R289Q/I336V, T49D/S59A/I336V,T49D/S59A/S521D/N531D, T49D/Q111K, T49D/Q111K/H234Y,T49D/Q111K/H234Y/R289Q/I336V/E523D/N531D/G549D,T49D/Q111K/H234Y/R289Q/S521D/E523D/N531D/G549D/I562S,T49D/Q111K/H234Y/R289Q/E523D/G549D, T49D/Q111K/H234Y/I336V/S521D/E523D,T49D/Q111K/H234Y/I336V/E523D/N531D, T49D/Q111K/H234Y/E523D/N531D/G549D,T49D/Q111K/H234Y/N531D, T49D/Q111K/H234Y/G549D, T49D/Q111K/R289Q,T49D/Q111K/R289Q/I336V/S521D/G549D, T49D/Q111K/E523D/N531D/G549D,T49D/Q111K/N531D/G549D, T49D/H234Y, T49D/H234Y/R289Q,T49D/H234Y/R289Q/I336V/N531D/G549D, T49D/H234Y/R289Q/E523D,T49D/H234Y/R289Q/E523D/N531D/A540R, T49D/H234Y/R289Q/E523D/G549D,T49D/H234Y/Q305E/G549D, T49D/H234Y/S521D, T49D/H234Y/G549D,T49D/R289Q/Q305E/I336V/E523D, T49D/R289Q/I336V,T49D/R289Q/I336V/S521D/N531D, T49D/I336V/S521D/I562S,T49D/S521D/N531D/G549D/I562S, T49D/S521D/G549D, T49D/E523D/G549D,T49D/G549D, S59A, S59A/Q111K/H234Y/R289Q/Q305E/I336V/G549D,S59A/Q111K/H234Y/R289Q/Q305E/G549D, S59A/Q111K/R289Q, S59A/Q111K/I336V,S59A/G549D, R103L, R103L/I336V/A519P/S548K, R103L/S521D/E523D/A540R,Q111K/H234Y/R289Q, Q111K/H234Y/R289Q/E523D, Q111K/R289Q/I336V/S521D,Q111K/R289Q/I336V/E523D/G549D, Q111K/I336V/I562S,Q111K/S521D/E523D/G549D, H234Y, H234Y/R289Q, H234Y/R289Q/I336V/E523D,H234Y/R289Q/E523D/N531D/G549D/I562S, H234Y/R289Q/E523D/G549D,H234Y/R289Q/N531D, H234Y/R289Q/G549D, H234Y/I336V,H234Y/I336V/N531D/I562S, H234Y/S521D/E523D, H234Y/S521D/E523D/G549D,H234Y/E523D/N531D, H234Y/N531D/G549D, H234Y/A540R, H234Y/G549D,H234Y/I562S, R289Q, R289Q/I336V/G549D, R289Q/S521D/E523D,R289Q/S521D/E523D/A540R, R289Q/S521D/E523D/G549D, R289Q/E523D,Q305E/I336V/L541A/S548K, I336V/S548K, A519P/S548K, S521D, S521D/S548K,S521D/G549D, S521D/I562S, E523D, N531D/G549D, A540R, and G549D, whereinthe positions are numbered with reference to SEQ ID NO: 736.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 24/44/464, 24/201/202/351/507, 24/202/351,33/37/46/56/103/523/540/548/549, 33/37/46/56/111/234/523,33/37/46/56/111/523/540, 33/37/46/103/111/234, 33/37/46/103/111/548/549,33/37/46/103/523, 33/37/46/111/234/328/523/540/548,33/37/46/111/336/540, 33/37/46/234/523, 33/37/46/336, 33/37/46/548/549,33/37/56/111/234/523/540/548, 33/37/56/234/523/540/548,33/37/103/111/234/336/540/548, 33/37/103/336/523/540/548,33/37/111/234/523/540/548, 33/37/111/523/540, 33/37/111/548,33/37/234/336/523/548, 33/37/523/540/548, 33/46, 33/46/56/111/548,33/46/56/234/248/549, 33/46/103/523/549, 33/56/103/111, 33/103/111/234,33/103/111/234/336/523/548, 33/103/111/234/540/549, 33/103/111/336,33/111/234/548, 33/234/328/523/540/549, 33/234/336/523/540/548/549,33/234/523/540/548, 33/234/523/548, 33/234/540/548/549, 33/336,33/336/523/540/548/549, 33/336/540/548, 33/336/549, 37, 37/46/56/523,37/46/234, 37/46/336/523/540/548, 37/46/336/540/548,37/56/234/336/523/540/548/549, 37/56/336/523/540/548,37/103/336/548/549, 37/111/523, 37/234/328/336/523, 37/336, 37/548/549,44, 46/103/328/336/523, 46/103/336, 46/234/336, 46/234/540/548,46/336/540/549, 202, 202/332/408, 234/523/540/548/549, 336/523, 408, and523/548, wherein the positions are numbered with reference to SEQ ID NO:790. In some embodiments, the tyrosine ammonia lyase comprises at leastone substitution or substitution sets selected from 24T/44R/464Q,24T/201N/202R/351N/507R, 24T/202R/351N,33N/37A/46R/56G/103L/523D/540R/548K/549D,33N/37A/46R/56G/111K/234Y/523D, 33N/37A/46R/56G/111K/523D/540R,33N/37A/46R/103L/111K/234Y, 33N/37A/46R/103L/111K/548K/549D,33N/37A/46R/103L/523D, 33N/37A/46R/111K/234Y/328Q/523D/540R/548K,33N/37A/46R/111K/336V/540R, 33N/37A/46R/234Y/523D, 33N/37A/46R/336V,33N/37A/46R/548K/549D, 33N/37A/56G/111K/234Y/523D/540R/548K,33N/37A/56G/234Y/523D/540R/548K, 33N/37A/103L/111K/234Y/336V/540R/548K,33N/37A/103L/336V/523D/540R/548K, 33N/37A/111K/234Y/523D/540R/548K,33N/37A/111K/523D/540R, 33N/37A/111K/548K, 33N/37A/234Y/336V/523D/548K,33N/37A/523D/540R/548K, 33N/46R, 33N/46R/56G/111K/548K,33N/46R/56G/234Y/248V/549D, 33N/46R/103L/523D/549D, 33N/56G/103L/111K,33N/103L/111K/234Y, 33N/103L/111K/234Y/336V/523D/548K,33N/103L/111K/234Y/540R/549D, 33N/103L/111K/336V, 33N/111K/234Y/548K,33N/234Y/328Q/523D/540R/549D, 33N/234Y/336V/523D/540R/548K/549D,33N/234Y/523D/540R/548K, 33N/234Y/523D/548K, 33N/234Y/540R/548K/549D,33N/336V, 33N/336V/523D/540R/548K/549D, 33N/336V/540R/548K,33N/336V/549D, 37A, 37A/46R/56G/523D, 37A/46R/234Y,37A/46R/336V/523D/540R/548K, 37A/46R/336V/540R/548K,37A/56G/234Y/336V/523D/540R/548K/549D, 37A/56G/336V/523D/540R/548K,37A/103L/336V/548K/549D, 37A/111K/523D, 37A/234Y/328Q/336V/523D,37A/336V, 37A/548K/549D, 44R, 46R/103L/328Q/336V/523D, 46R/103L/336V,46R/234Y/336V, 46R/234Y/540R/548K, 46R/336V/540R/549D, 202R,202R/332Q/408E, 234Y/523D/540R/548K/549D, 336V/523D, 408E, and523D/548K, wherein the positions are numbered with reference to SEQ IDNO: 790. In some embodiments, the tyrosine ammonia lyase comprises atleast one substitution or substitution sets selected fromI24T/K44R/R464Q, I24T/K201N/L202R/G351N/Q507R, I24T/L202R/G351N,K33N/Q37A/L46R/R56G/R103L/E523D/A540R/S548K/G549D,K33N/Q37A/L46R/R56G/Q111K/H234Y/E523D,K33N/Q37A/L46R/R56G/Q111K/E523D/A540R, K33N/Q37A/L46R/R103L/Q111K/H234Y,K33N/Q37A/L46R/R103L/Q111K/S548K/G549D, K33N/Q37A/L46R/R103L/E523D,K33N/Q37A/L46R/Q111K/H234Y/A328Q/E523D/A540R/S548K,K33N/Q37A/L46R/Q111K/I336V/A540R, K33N/Q37A/L46R/H234Y/E523D,K33N/Q37A/L46R/I336V, K33N/Q37A/L46R/S548K/G549D,K33N/Q37A/R56G/Q111K/H234Y/E523D/A540R/S548K,K33N/Q37A/R56G/H234Y/E523D/A540R/S548K,K33N/Q37A/R103L/Q111K/H234Y/I336V/A540R/S548K,K33N/Q37A/R103L/I336V/E523D/A540R/S548K,K33N/Q37A/Q111K/H234Y/E523D/A540R/S548K, K33N/Q37A/Q111K/E523D/A540R,K33N/Q37A/Q111K/S548K, K33N/Q37A/H234Y/I336V/E523D/S548K,K33N/Q37A/E523D/A540R/S548K, K33N/L46R, K33N/L46R/R56G/Q111K/S548K,K33N/L46R/R56G/H234Y/A248V/G549D, K33N/L46R/R103L/E523D/G549D,K33N/R56G/R103L/Q111K, K33N/R103L/Q111K/H234Y,K33N/R103L/Q111K/H234Y/I336V/E523D/S548K,K33N/R103L/Q111K/H234Y/A540R/G549D, K33N/R103L/Q111K/I336V,K33N/Q111K/H234Y/S548K, K33N/H234Y/A328Q/E523D/A540R/G549D,K33N/H234Y/I336V/E523D/A540R/S548K/G549D, K33N/H234Y/E523D/A540R/S548K,K33N/H234Y/E523D/S548K, K33N/H234Y/A540R/S548K/G549D, K33N/I336V,K33N/I336V/E523D/A540R/S548K/G549D, K33N/I336V/A540R/S548K,K33N/I336V/G549D, Q37A, Q37A/L46R/R56G/E523D, Q37A/L46R/H234Y,Q37A/L46R/I336V/E523D/A540R/S548K, Q37A/L46R/I336V/A540R/S548K,Q37A/R56G/H234Y/1336V/E523D/A540R/5548K/G549D,Q37A/R56G/1336V/E523D/A540R/S548K, Q37A/R103L/1336V/S548K/G549D,Q37A/Q111K/E523D, Q37A/H234Y/A328Q/I336V/E523D, Q37A/I336V,Q37A/S548K/G549D, K44R, L46R/R103L/A328Q/1336V/E523D, L46R/R103L/1336V,L46R/H234Y/I336V, L46R/H234Y/A540R/5548K, L46R/I336V/A540R/G549D, L202R,L202R/K332Q/D408E, H234Y/E523D/A540R/S548K/G549D, I336V/E523D, D408E,and E523D/S548K, wherein the positions are numbered with reference toSEQ ID NO: 790.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 74, 102, 210, 313, 391, 392, 402, 415, 417, 418, 420, 424,506, and 530, wherein the positions are numbered with reference to SEQID NO: 790. In some embodiments, the tyrosine ammonia lyase comprises atleast one substitution or substitution sets selected from 74A, 102P,210T, 313I, 313M, 391N, 392A, 402A, 415F, 417A, 418V, 420L, 424A, 506C,and 530V, wherein the positions are numbered with reference to SEQ IDNO: 790. In some embodiments, the tyrosine ammonia lyase comprises atleast one substitution or substitution sets selected from I74A, M102P,L210T, L313I, L313M, T391N, M392A, S402A, L415F, G417A, L418V, I420L,S424A, R506C, and D530V, wherein the positions are numbered withreference to SEQ ID NO: 790.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 24, 24/44, 24/44/46/202/391, 24/44/111/202/464/506,24/44/202/313/391/540, 24/44/549, 24/46/102/313/506,24/46/103/111/464/506/549, 24/46/103/391/506, 24/46/111/313/391,24/46/111/313/549, 24/46/111/506, 24/46/202/391/506, 24/46/549,24/103/202, 24/202, 44/46/102/202/313/417, 44/46/103/111/391,44/46/103/202/313/391, 44/46/103/202/417/464, 44/103/111/313/391/540,44/111/464/506, 46/103/506, 46/111/417, 46/111/506, 46/202,46/202/506/549, 46/313/391/540, 103/313/549, 111, 111/202,111/202/313/391/464, 111/202/313/391/506, 111/202/313/417/540/549,111/202/391, 111/202/391/540, 111/464/540/549, 202/313/391/549,202/417/464/540/549, 202/464/506, 202/506/540, 202/506/540/549,313/391/540/549, and 417, wherein the positions are numbered withreference to SEQ ID NO: 1454. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom 24T, 24T/44R, 24T/44R/46R/202R/391N, 24T/44R/111K/202R/464Q/506C,24T/44R/202R/313M/391N/540R, 24T/44R/549D, 24T/46R/102P/313M/506C,24T/46R/103L/1111K/464Q/506C/549D, 24T/46R/103L/391N/506C,24T/46R/111K/313M/391N, 24T/46R/111K/313M/549D, 24T/46R/111K/506C,24T/46R/202R/391N/506C, 24T/46R/549D, 24T/103L/202R, 24T/202R,44R/46R/102P/202R/313M/417A, 44R/46R/103L/1111K/391N,44R/46R/103L/202R/313M/391N, 44R/46R/103L/202R/417A/464Q,44R/103L/111K/313M/391N/540R, 44R/111K/464Q/506C, 46R/103L/506C,46R/111K/417A, 46R/111K/506C, 46R/202R, 46R/202R/506C/549D,46R/313M/391N/540R, 103L/313M/549D, 111K, 111K/202R,111K/202R/313M/391N/464Q, 111K/202R/313M/391N/506C,111K/202R/313M/417A/540R/549D, 111K/202R/391N, 111K/202R/391N/540R,111K/464Q/540R/549D, 202R/313M/391N/549D, 202R/417A/464Q/540R/549D,202R/464Q/506C, 202R/506C/540R, 202R/506C/540R/549D,313M/391N/540R/549D, and 417A, wherein the positions are numbered withreference to SEQ ID NO: 1454. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom I24T, I24T/K44R, I24T/K44R/L46R/L202R/T391N,I24T/K44R/Q111K/L202R/R464Q/R506C, I24T/K44R/L202R/L313M/T391N/A540R,I24T/K44R/G549D, I24T/L46R/M102P/L313M/R506C,I24T/L46R/R103L/Q111K/R464Q/R506C/G549D, I24T/L46R/R103L/T391N/R506C,I24T/L46R/Q111K/L313M/T391N, I24T/L46R/Q111K/L313M/G549D,I24T/L46R/Q111K/R506C, I24T/L46R/L202R/T391N/R506C, I24T/L46R/G549D,I24T/R103L/L202R, I24T/L202R, K44R/L46R/M102P/L202R/L313M/G417A,K44R/L46R/R103L/Q111K/T391N, K44R/L46R/R103L/L202R/L313M/T391N,K44R/L46R/R103L/L202R/G417A/R464Q, K44R/R103L/Q111K/L313M/T391N/A540R,K44R/Q111K/R464Q/R506C, L46R/R103L/R506C, L46R/Q111K/G417A,L46R/Q111K/R506C, L46R/L202R, L46R/L202R/R506C/G549D,L46R/L313M/T391N/A540R, R103L/L313M/G549D, Q111K, Q111K/L202R,Q111K/L202R/L313M/T391N/R464Q, Q111K/L202R/L313M/T391N/R506C,Q111K/L202R/L313M/G417A/A540R/G549D, Q111K/L202R/T391N,Q111K/L202R/T391N/A540R, Q111K/R464Q/A540R/G549D,L202R/L313M/T391N/G549D, L202R/G417A/R464Q/A540R/G549D,L202R/R464Q/R506C, L202R/R506C/A540R, L202R/R506C/A540R/G549D,L313M/T391N/A540R/G549D, and G417A, wherein the positions are numberedwith reference to SEQ ID NO: 1454.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 20/44/202/506, 20/103/111/471/506, 20/202/313/471/506, 24,27, 33, 36, 41, 44, 44/111/202/464/471/506/549, 44/202/464/506, 46, 48,50, 51, 55, 56/111/506, 56/506, 59, 66, 66/528, 103/111/202/464/471,103/111/471, 103/202/540, 103/464/549, 103/471, 111, 111/202/313/316,111/202/313/471/506, 111/202/471, 111/506, 197, 202,202/313/464/471/506, 202/464/506, 202/464/506/549, 208, 272, 289,292/553, 305, 312/559, 313/464/471/506, 331, 332, 432, 435, 464,464/506, 470, 500, 504, 506, 506/549, 518, 519, 523, 540, 543, 547,547/553, 555, 556, and 559, wherein the positions are numbered withreference to SEQ ID NO: 1578. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom 20G/44R/202R/506R, 20G/103L/111K/471Y/506R,20G/202R/313L/471Y/506R, 24E, 24K, 24Q, 24S, 27S, 33E, 36S, 36T, 41A,41H, 41L, 41N, 41Q, 41S, 44E, 44P, 44R,44R/111K/202R/464Q/471Y/506R/549D, 44R/202R/464Q/506R, 44S, 46A, 46S,48A, 48G, 50M, 51A, 51H, 55S, 56G/111K/506R, 56G/506R, 59N, 59S,66H/528R, 66K, 66N, 66T, 103L/111K/202R/464Q/471Y, 103L/111K/471Y,103L/202R/540R, 103L/464Q/549D, 103L/471Y, 111K, 111K/202R/313L/316Q,111K/202R/313L/471Y/506R, 111K/202R/471Y, 111K/506R, 197V, 202R,202R/313L/464Q/471Y/506R, 202R/464Q/506R, 202R/464Q/506R/549D, 208M,272A, 289L, 292L/553S, 305D, 305E, 312C/559Y, 313L/464Q/471Y/506R, 331G,332N, 432L, 432T, 435A, 435T, 464N, 464Q/506R, 464S, 470I, 470T, 500L,504A, 504L, 506E, 506R, 506R/549D, 518S, 519E, 519K, 519P, 519T, 523H,540H, 540R, 543T, 543V, 547G, 547M/553I, 555S, 556L, 556V, 559C, 559H,559I, 559L, 559M, and 559Y, wherein the positions are numbered withreference to SEQ ID NO: 1578. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom D20G/K44R/L202R/C506R, D20G/R103L/Q111K/Q471Y/C506R,D20G/L202R/M313L/Q471Y/C506R, T24E, T24K, T24Q, T24S, D27S, N33E, A36S,A36T, Y41A, Y41H, Y41L, Y41N, Y41Q, Y41S, K44E, K44P, K44R,K44R/Q111K/L202R/R464Q/Q471Y/C506R/G549D, K44R/L202R/R464Q/C506R, K445,R46A, R46S, T48A, T48G, K50M, E51A, E51H, Q55S, R56G/Q111K/C506R,R56G/C506R, A59N, A59S, Q66H/W528R, Q66K, Q66N, Q66T,R103L/Q111K/L202R/R464Q/Q471Y, R103L/Q111K/Q471Y, R103L/L202R/A540R,R103L/R464Q/G549D, R103L/Q471Y, Q111K, Q111K/L202R/M313L/L316Q,Q111K/L202R/M313L/Q471Y/C506R, Q111K/L202R/Q471Y, Q111K/C506R, P197V,L202R, L202R/M313L/R464Q/Q471Y/C506R, L202R/R464Q/C506R,L202R/R464Q/C506R/G549D, L208M, P272A, Q289L, R292L/V553S, Q305D, Q305E,S312C/V559Y, M313L/R464Q/Q471Y/C506R, A331G, K332N, Y432L, Y432T, S435A,S435T, R464N, R464Q/C506R, R464S, Q470I, Q470T, C500L, R504A, R504L,C506E, C506R, C506R/G549D, K518S, A519E, A519K, A519P, A519T, D523H,A540H, A540R, A543T, A543V, T547G, T547M/V553I, A555S, I556L, I556V,V559C, V559H, V559I, V559L, V559M, and V559Y, wherein the positions arenumbered with reference to SEQ ID NO: 1578.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 20, 20/44/208/272/519, 20/111/208/272/410/432,20/197/432/519, 24, 24/46/410/435/518, 36/55/305/332/410,36/66/197/305/332/457, 36/66/197/305/410, 36/66/305, 36/66/305/332/410,36/66/305/500, 36/197/305, 36/197/305/332/410, 36/197/305/332/410/500,36/197/305/332/410/559, 36/197/305/332/500, 36/197/305/410,36/197/305/500, 36/305, 36/305/332, 36/305/332/410, 36/305/410,41/44/46/111/272, 41/432/526, 44/46/111/208, 44/46/111/432/519,44/111/432/519/526, 44/208/432/435, 46/111/208/272/435/526,46/208/272/432/435/526, 46/432/471, 55/305, 55/305/332/457/543/547,55/305/500/559, 66/305, 66/305/332, 111, 111/272/432/435,111/272/471/526, 111/272/519, 111/432/435, 111/432/526, 111/526,134/202/305, 197/305, 197/305/332/410, 197/305/332/500/543/547,197/305/410, 197/305/500, 208, 208/272/432, 208/410/435, 208/435/519,211/410, 272, 272/435/526, 298/410, 305, 305/332, 305/332/410,305/332/410/500, 305/332/457, 305/332/457/500, 305/332/457/555,305/332/500, 305/410, 305/500/543/547, 410, 432/526, 435, 435/518,435/519/526, 471, 471/526, and 519, wherein the positions are numberedwith reference to SEQ ID NO: 1660. In some embodiments, the tyrosineammonia lyase comprises at least one substitution or substitution setsselected from 20G, 20G/44E/208M/272A/519K, 20G/111K/208M/272A/410M/432T,20G/197V/432T/519K, 24E, 24E/46S/410M/435T/518S, 36S/55S/305D/332N/410M,36S/66N/197V/305D/332N/457S, 36S/66N/197V/305D/410M, 36S/66N/305D,36S/66N/305D/332N/410M, 36S/66N/305D/500L, 36S/197V/305D,36S/197V/305D/332N/410M, 36S/197V/305D/332N/410M/500L,36S/197V/305D/332N/410M/559I, 36S/197V/305D/332N/500L,36S/197V/305D/410M, 36S/197V/305D/500L, 36S/305D, 36S/305D/332N,36S/305D/332N/410M, 36S/305D/410M, 41L/44E/46S/111K/272A, 41L/432T/526L,44E/46S/111K/208M, 44E/46S/111K/432T/519K, 44E/111K/432L/519K/526L,44E/208M/432T/435T, 46S/111K/208M/272A/435T/526L,46S/208M/272A/432L/435T/526L, 46S/432L/471Y, 55S/305D,55S/305D/332N/457S/543V/547G, 55S/305D/500L/559I, 66N/305D,66N/305D/332N, 111K, 111K/272A/432T/435T, 111K/272A/471Y/526L,111K/272A/519K, 111K/432L/435T, 111K/432T/526L, 111K/526L,134C/202L/305D, 197V/305D, 197V/305D/332N/410M,197V/305D/332N/500L/543V/547G, 197V/305D/410M, 197V/305D/500L, 208M,208M/272A/432L, 208M/410M/435T, 208M/435T/519K, 211Q/410I, 272A,272A/435T/526L, 298G/410I, 305D, 305D/332N, 305D/332N/410M,305D/332N/410M/500L, 305D/332N/457S, 305D/332N/457S/500L,305D/332N/457S/555S, 305D/332N/500L, 305D/410M, 305D/500L/543V/547G,410L, 432L/526L, 435T, 435T/518S, 435T/519K/526L, 471Y, 471Y/526L, and519K, wherein the positions are numbered with reference to SEQ ID NO:1660. In some embodiments, the tyrosine ammonia lyase comprises at leastone substitution or substitution sets selected from D20G,D20G/K44E/L208M/P272A/A519K, D20G/Q111K/L208M/P272A/P410M/Y432T,D20G/P197V/Y432T/A519K, T24E, T24E/R46S/P410M/S435T/K518S,A36S/Q55S/Q305D/K332N/P410M, A36S/Q66N/P197V/Q305D/K332N/G457S,A36S/Q66N/P197V/Q305D/P410M, A36S/Q66N/Q305D,A36S/Q66N/Q305D/K332N/P410M, A36S/Q66N/Q305D/C500L, A36S/P197V/Q305D,A36S/P197V/Q305D/K332N/P410M, A36S/P197V/Q305D/K332N/P410M/C500L,A36S/P197V/Q305D/K332N/P410M/V559I, A36S/P197V/Q305D/K332N/C500L,A36S/P197V/Q305D/P410M, A36S/P197V/Q305D/C500L, A36S/Q305D,A36S/Q305D/K332N, A36S/Q305D/K332N/P410M, A36S/Q305D/P410M,Y41L/K44E/R46S/Q111K/P272A, Y41L/Y432T/Y526L, K44E/R46S/Q111K/L208M,K44E/R46S/Q111K/Y432T/A519K, K44E/Q111K/Y432L/A519K/Y526L,K44E/L208M/Y432T/S435T, R46S/Q111K/L208M/P272A/S435T/Y526L,R46S/L208M/P272A/Y432L/S435T/Y526L, R46S/Y432L/Q471Y, Q55S/Q305D,Q55S/Q305D/K332N/G457S/A543V/T547G, Q55S/Q305D/C500L/V559I, Q66N/Q305D,Q66N/Q305D/K332N, Q111K, Q111K/P272A/Y432T/S435T,Q111K/P272A/Q471Y/Y526L, Q111K/P272A/A519K, Q111K/Y432L/S435T,Q111K/Y432T/Y526L, Q111K/Y526L, S134C/R202L/Q305D, P197V/Q305D,P197V/Q305D/K332N/P410M, P197V/Q305D/K332N/C500L/A543V/T547G,P197V/Q305D/P410M, P197V/Q305D/C500L, L208M, L208M/P272A/Y432L,L208M/P410M/S435T, L208M/S435T/A519K, L211Q/P410I, P272A,P272A/S435T/Y526L, S298G/P410I, Q305D, Q305D/K332N, Q305D/K332N/P410M,Q305D/K332N/P410M/C500L, Q305D/K332N/G457S, Q305D/K332N/G457S/C500L,Q305D/K332N/G457S/A555S, Q305D/K332N/C500L, Q305D/P410M,Q305D/C500L/A543V/T547G, P410L, Y432L/Y526L, S435T, S435T/K518S,S435T/A519K/Y526L, Q471Y, Q471Y/Y526L, and A519K, wherein the positionsare numbered with reference to SEQ ID NO: 1660.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 24/59/111/289/504, 24/111/289, 24/111/289/500/504,24/111/432, 24/111/435/500, 24/289, 24/432/435/504, 41/44/208/547,41/51/410/543, 41/208/410/543/547, 41/208/518/519/547, 41/208/547,41/410/518/543, 41/410/518/547, 41/543, 44/208/518/547, 51/519,59/66/111/289/432/500, 59/66/111/432/500, 59/66/111/432/504,59/66/289/500/504/556, 59/111, 59/111/432/500, 59/289, 59/289/435,59/432, 59/432/435/504/556, 66/111, 111, 111/289, 111/289/435,111/289/435/500, 111/289/500, 111/331/432/504, 111/331/435/500, 111/432,111/432/500, 111/500/504, 208, 208/211/410/519, 208/211/547, 211,211/518/519/547, 211/547, 289, 289/432, 289/432/435, 289/435,289/435/500/504, 289/435/504/556, 289/504, 410, 432, 435/504, 504,504/556, 519, and 547, wherein the positions are numbered with referenceto SEQ ID NO: 1844.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from 24K/59S/111K/289L/504A,24K/111K/289L, 24K/111K/289L/500L/504A, 24K/111K/432L,24K/111K/435T/500L, 24K/289L, 24K/432L/435T/504A, 41Q/44P/208M/547G,41Q/51A/410L/543V, 41Q/208M/410L/543V/547G, 41Q/208M/518S/519T/547G,41Q/208M/547G, 41Q/410L/518S/543V, 41Q/410L/518S/547G, 41Q/543V,44R/208M/518S/547G, 51A/519T, 59S/66T/111K/289L/432L/500L,595/66T/111K/432L/500L, 59S/66T/111K/432L/504A,59S/66T/289L/500L/504A/556L, 59S/111K, 59S/111K/432L/500L, 59S/289L,59S/289L/435T, 59S/432L, 59S/432L/435T/504A/556L, 66T/111K, 111K,111K/289L, 111K/289L/435T, 111K/289L/435T/500L, 111K/289L/500L,111K/331G/432L/504A, 111K/331G/435T/500L, 111K/432L, 111K/432L/500L,111K/500L/504A, 208M, 208M/211Q/410L/519T, 208M/211Q/547G, 211Q,211Q/518S/519T/547G, 211Q/547G, 289L, 289L/432L, 289L/432L/435T,289L/435T, 289L/435T/500L/504A, 289L/435T/504A/556L, 289L/504A, 410L,432L, 435T/504A, 504A, 504A/556L, 519T, and 547G, wherein the positionsare numbered with reference to SEQ ID NO: 1844. In some embodiments, thetyrosine ammonia lyase comprises at least one substitution orsubstitution sets selected from T24K/A59S/Q111K/Q289L/R504A,T24K/Q111K/Q289L, T24K/Q111K/Q289L/C500L/R504A, T24K/Q111K/Y432L,T24K/Q111K/S435T/C500L, T24K/Q289L, T24K/Y432L/S435T/R504A,Y41Q/K44P/L208M/T547G, Y41Q/E51A/M410L/A543V,Y41Q/L208M/M410L/A543V/T547G, Y41Q/L208M/K518S/A519T/T547G,Y41Q/L208M/T547G, Y41Q/M410L/K518S/A543V, Y41Q/M410L/K518S/T547G,Y41Q/A543V, K44R/L208M/K518S/T547G, E51A/A519T,A59S/Q66T/Q111K/Q289L/Y432L/C500L, A59S/Q66T/Q111K/Y432L/C500L,A59S/Q66T/Q111K/Y432L/R504A, A59S/Q66T/Q289L/C500L/R504A/I556L,A59S/Q111K, A59S/Q111K/Y432L/C500L, A59S/Q289L, A59S/Q289L/S435T,A59S/Y432L, A59S/Y432L/S435T/R504A/I556L, Q66T/Q111K, Q111K,Q111K/Q289L, Q111K/Q289L/S435T, Q111K/Q289L/S435T/C500L,Q111K/Q289L/C500L, Q111K/A331G/Y432L/R504A, Q111K/A331G/S435T/C500L,Q111K/Y432L, Q111K/Y432L/C500L, Q111K/C500L/R504A, L208M,L208M/L211Q/M410L/A519T, L208M/L211Q/T547G, L211Q,L211Q/K518S/A519T/T547G, L211Q/T547G, Q289L, Q289L/Y432L,Q289L/Y432L/S435T, Q289L/S435T, Q289L/S435T/C500L/R504A,Q289L/S435T/R504A/I556L, Q289L/R504A, M410L, Y432L, S435T/R504A, R504A,R504A/1556L, A519T, and T547G, wherein the positions are numbered withreference to SEQ ID NO: 1844.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 4, 8, 11, 13, 13/22/45/512, 13/45, 13/45/512, 17, 19, 45,45/244/324/513, 45/436/512/513, 45/512, 144, 251, 324, 327, 341, and428, wherein the positions are numbered with reference to SEQ ID NO:2030. In some embodiments, the tyrosine ammonia lyase comprises at leastone substitution or substitution sets selected from 4D, 8D, 8G, 8R, 11A,11D, 11G, 11K, 11S, 13Q/45V/512V, 13R, 13S/22T/45V/512V, 13S/45V, 17Q,19H, 45V, 45V/244M/324V/513R, 45V/436I/512V/513R, 45V/512V, 144L, 251V,324R, 327M, 341A, and 428V, wherein the positions are numbered withreference to SEQ ID NO: 2030. In some embodiments, the tyrosine ammonialyase comprises at least one substitution or substitution sets selectedfrom A4D, Q8D, Q8G, Q8R, T11A, T11D, T11G, T11K, T11S, K13Q/A45V/I512V,K13R, K13S/V22T/A45V/I512V, K13S/A45V, T17Q, S19H, A45V,A45V/L244M/A324V/K513R, A45V/L436I/I512V/K513R, A45V/I512V, I144L,L251V, A324R, I327M, V341A, and L428V, wherein the positions arenumbered with reference to SEQ ID NO: 2030.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 2, 4, 4/8/11/13/22/111/504, 4/8/11/17/324/428/504,4/8/11/22/111, 4/8/22/111/504, 4/17/241/519/547, 4/324, 4/428, 6, 7,8/11/13/324, 8/11/17/22/324/504/519, 8/11/111/504, 10, 11/13/504,11/17/22/324, 14, 16, 39, 57, 228, 228/259, 236, 305/307/425, 316, 385,425, 430, 437, 478, and 505, wherein the positions are numbered withreference to SEQ ID NO: 2114.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from 2G, 2T, 4D,4D/8D/11G/17P/324V/428V/504A, 4D/8D/22T/111K/504A,4D/8R/11D/13R/22T/111K/504A, 4D/8R/11D/22T/111K, 4D/17Q/241S/519T/547A,4D/324V, 4D/428V, 6G, 7Q, 8D/11D/13R/324V,8D/11D/17Q/22T/324V/504A/519T, 8D/11D/111K/504A, 10G, 11D/17Q/22T/324V,11G/13R/504A, 14G, 16Q, 16T, 39S, 57I, 228M, 228M/259H, 236V,305E/307N/425V, 316T, 385A, 425V, 430E, 437G, 478L, and 505S, whereinthe positions are numbered with reference to SEQ ID NO: 2114. In someembodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from I2G, I2T, A4D,A4D/Q8D/T11G/T17P/A324V/L428V/R504A, A4D/Q8D/V22T/Q111K/R504A,A4D/Q8R/T11D/K13R/V22T/Q111K/R504A, A4D/Q8R/T11D/V22T/Q111K,A4D/T17Q/A241S/A519T/T547A, A4D/A324V, A4D/L428V, Q6G, T7Q,Q8D/T11D/K13R/A324V, Q8D/T11D/T17Q/V22T/A324V/R504A/A519T,Q8D/T11D/Q111K/R504A, F10G, T11D/T17Q/V22T/A324V, T11G/K13R/R504A, S14G,H16Q, H16T, A39S, V57I, I228M, I228M/Q259H, A236V, D305E/I307N/I425V,L316T, V385A, I425V, T430E, T437G, M478L, and T505S, wherein thepositions are numbered with reference to SEQ ID NO: 2114.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution set at one or more amino acid positionsselected from 4/7/57/111, 4/7/111/114/437/547, 4/16/385/478,4/57/111/437, 7/385/547, 10/16/437, 16, 16/41/57/111/385/547,16/41/57/437/478, 16/41/437, 16/57, 16/241/437, 16/385, 16/385/437,16/385/478/547, 16/437, 20/385/437/478/547, 57, 57/111/114,57/111/478/547, 57/437,111/114/202/234/289/305/313/324/332/336/410/432/435/464/504/512/519/523/548/549,111/241/437/478, 111/478, 241, 241/385, 241/385/478, 241/385/478/547,241/437, 385, 385/437, 385/437/478/547, 437, 437/478, and 478, whereinthe positions are numbered with reference to SEQ ID NO: 2156.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from 4D/7Q/57I/111K,4D/7Q/111K/114S/437G/547G, 4D/16Q/385A/478L, 4D/57I/111K/437G,7Q/385A/547G, 10G/16Q/437G, 16Q, 16Q/41Q/57I/111K/385A/547G,16Q/41Q/57I/437G/478L, 16Q/41Q/437G, 16Q/57I, 16Q/241S/437G, 16Q/385A,16Q/385A/437G, 16Q/385A/478L/547G, 16Q/437G, 20G/385A/437G/478L/547G,57I, 57I/111K/114S, 57I/111K/478L/547G, 57I/437G,111K/114S/202L/234H/289Q/305Q/313L/324A/332K/336I/410P/432Y/435S/464R/504R/512I/519A/523E/548S/549G,111K/241S/437G/478L, 111K/478L, 241S, 241S/385A, 241S/385A/478L,241S/385A/478L/547G, 241S/437G, 385A, 385A/437G, 385A/437G/478L/547G,437G, 437G/478L, and 478L, wherein the positions are numbered withreference to SEQ ID NO: 2156.

In some embodiments, the tyrosine ammonia lyase comprises at least onesubstitution or substitution sets selected from A4D/T7Q/V57I/Q111K,A4D/T7Q/Q111K/P114S/T437G/T547G, A4D/H16Q/V385A/M478L,A4D/V57I/Q111K/T437G, T7Q/V385A/T547G, F10G/H16Q/T437G, H16Q,H16Q/Y41Q/V57I/Q111K/V385A/T547G, H16Q/Y41Q/V57I/T437G/M478L,H16Q/Y41Q/T437G, H16Q/V57I, H16Q/A241S/T437G, H16Q/V385A,H16Q/V385A/T437G, H16Q/V385A/M478L/T547G, H16Q/T437G,D20G/V385A/T437G/M478L/T547G, V57I, V57I/Q111K/P114S,V57I/Q111K/M478L/T547G, V57I/T437G,Q111K/P114S/R202L/Y234H/L289Q/D305Q/M313L/V324A/N332K/V336I/M410P/L432Y/T435S/Q464R/A504RN512I/T519A/D523E/K548S/D549G,Q111K/A241S/T437G/M478L, Q111K/M478L, A241S, A241S/V385A,A241S/V385A/M478L, A241S/V385A/M478L/T547G, A241S/T437G, V385A,V385A/T437G, V385A/T437G/M478L/T547G, T437G, T437G/M478L, and M478L,wherein the positions are numbered with reference to SEQ ID NO: 2156.

The present invention also provides the variants provided in Tables 1-1,2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 9-2, 10-1, 10-2, 11-1, 12-1,13-1, 14-1, 15-1, 16-1, and/or 17-1. In some embodiments, the variantcomprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments,the recombinant tyrosine lyase variant comprises at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQID NO: 2. In some additional embodiments, the recombinant tyrosine lyasevariant comprises at least 90% sequence identity to at least one of theeven numbered sequences in SEQ ID NOS: 10-2290. In some additionalembodiments, the polypeptide of the recombinant tyrosine ammonia lyasevariant comprises one of the even-numbered sequences of SEQ ID NOS:10-2290. In some further embodiments, the recombinant tyrosine ammonialyase comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptidesequence of SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578,1660, 1844, 2030, 2114, and/or 2156.

In some additional embodiments, the recombinant tyrosine ammonia lyaseprovided herein is thermostable. In some further embodiments, therecombinant tyrosine ammonia lyase is resistant to proteolysis. In yetsome additional embodiments, the recombinant tyrosine ammonia lyase isresistant to at least one digestive tract protease. In some embodiments,the digestive tract protease is selected from chymotrypsin, trypsin,carboxypeptidases, and elastases. In some further embodiments, therecombinant tyrosine ammonia lyase is acid stable. In some additionalembodiments, the recombinant tyrosine ammonia lyase is acid stable andresistant to proteolysis. In yet some additional embodiments, therecombinant tyrosine ammonia lyase is purified. In some furtherembodiments, the recombinant tyrosine ammonia lyase exhibits at leastone improved property selected from: i) enhanced catalytic activity; ii)reduced sensitivity to proteolysis; iii) increased tolerance to acidicpH; iv) reduced aggregation; v) decreased Km for tyrosine; vi) decreasedimmunogenicity; or a combination of any of i), ii), iii), iv), v),and/or vi), as compared to a reference sequence. In some embodiments,the reference sequence is selected from SEQ ID NO: 2, 14, 86, 334, 388,604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156.

The present invention also provides compositions comprising at least onerecombinant tyrosine ammonia lyase provided herein. In some embodiments,the compositions comprise one recombinant tyrosine ammonia lyaseprovided herein.

The present invention also provides recombinant polynucleotide sequencesencoding at least one recombinant tyrosine ammonia lyase providedherein. In some embodiment, a recombinant polynucleotide sequenceencodes one recombinant tyrosine ammonia lyase. In some embodiments, thepolynucleotide sequence is codon-optimized. In some additionalembodiments, the polynucleotide comprises at least 90% sequence identityto at least one of the odd-numbered sequences of SEQ ID NOS: 9-2289. Inyet some further embodiments, the recombinant polynucleotide sequencecomprises an odd-numbered sequence of SEQ ID NOS: 9-2289.

The present invention also provides expression vectors comprising atleast one recombinant polynucleotide sequence encoding a recombinanttyrosine ammonia lyase provided herein. In some embodiments, theexpression vectors comprises a recombinant polynucleotide sequenceencoding a recombinant tyrosine ammonia lyase provided herein. In someembodiments of the expression vectors, the recombinant polynucleotidesequence is operably linked to a control sequence. In some additionalembodiments of the expression vectors, the control sequence comprises apromoter. In some additional embodiments of the expression vectors, thecontrol sequence is a promoter. In some embodiments of the expressionvectors, the promoter is a heterologous promoter.

The present invention also provides host cells comprising at least oneexpression vector provided herein. In some embodiments, the host cellcomprises one expression vector provided herein, while in somealternative embodiments, the host cell comprises two or more expressionvectors provided herein. In some embodiments, the host cell isprokaryotic, while in some alternative embodiments, the host cell iseukaryotic.

The present invention also provides methods of producing a recombinanttyrosine ammonia lyase, comprising culturing at least one host cellprovided herein, under conditions that the tyrosine ammonia lyaseencoded by the recombinant polynucleotide is produced. In someembodiments, the methods further comprise the step of recovering therecombinant tyrosine ammonia lyase. In some additional embodiments, themethods further comprise the step of purifying the recombinant tyrosineammonia lyase.

The present invention also provides pharmaceutical compositions for thetreatment of tyrosinemia comprising at least one recombinant tyrosineammonia lyase provided herein. In some embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrierand/or excipient. In some additional embodiments, the pharmaceuticalcomposition is suitable for oral administration to a human In someembodiments, the pharmaceutical composition is in the form of a pill,tablet, capsule, gelcap, liquid, or emulsion. In yet some furtherembodiments, the pharmaceutical composition is coadministered withnitisinone. In some additional embodiments, the pharmaceuticalcomposition comprises nitisinone. In some embodiments, the pill, tablet,capsule, or gelcap further comprises an enteric coating. In someadditional embodiments, the pharmaceutical composition is suitable forparenteral injection into a human. In yet some additional embodiments,the pharmaceutical composition is coadministered with a phenylalanineammonia lyase. In some further embodiments, the pharmaceuticalcomposition comprises phenylalanine ammonia lyase. In some embodiments,the pharmaceutical composition comprises a pill, tablet, capsule, orgelcap that further comprises an enteric coating. In yet some additionalembodiments, the pharmaceutical composition is coadministered with bothnitisinone and a phenylalanine ammonia lyase. In some furtherembodiments, the pharmaceutical composition comprises both nitisinoneand phenylalanine ammonia lyase. In some embodiments, the pharmaceuticalcomposition comprises a pill, tablet, capsule, or gelcap that furthercomprises an enteric coating.

The present invention also provides methods for treating and/orpreventing the symptoms of tyrosinemia or alkaptonuria in a subject,comprising providing a subject having tyrosinemia or alkaptonuria, andproviding at least one pharmaceutical composition provided herein to thesubject. In some embodiments, the methods comprise administering morethan one composition or pharmaceutical composition provided herein areadministered to the subject. In some alternative embodiments, only onecomposition or pharmaceutical composition is administered to thesubject. In some embodiments, the symptoms of tyrosinemia oralkaptonuria are ameliorated. In yet some further embodiments, thesubject is able to eat a diet that is less restricted in itsphenylalanine, methionine, and/or tyrosine content than diets requiredby subjects exhibiting the symptoms of tyrosinemia or alkaptonuria. Insome embodiments, the subject is an infant or child, while in somealternative embodiments, the subject is an adult or young adult.

The present invention also provides methods for the production ofL-tyrosine and/or L-tyrosine derivatives comprising the steps ofproviding at least one recombinant tyrosine ammonia lyase(s) providedherein and a suitable substrate, and combining the recombinant tyrosineammonia lyase(s) and the substrate under conditions such that L-tyrosineand/or at least one L-tyrosine derivative is produced. In someembodiments, only one recombinant tyrosine ammonia lyase is utilized inthe methods, while in some alternative embodiments, at least tworecombinant tyrosine ammonia lyases are utilized.

The present invention also provides methods for the production ofcoumaric acid, comprising the steps of providing at least onerecombinant tyrosine ammonia lyase, and a suitable substrate, andcombining the recombinant tyrosine ammonia lyase(s) and the substrateunder conditions such that coumaric acid is produced. In someembodiments, only one recombinant tyrosine ammonia lyase is utilized inthe methods, while in some alternative embodiments, at least tworecombinant tyrosine ammonia lyases are utilized.

The present invention also provides use of the compositions andpharmaceutical compositions provided herein.

DESCRIPTION OF THE INVENTION

The present invention provides engineered tyrosine ammonia-lyase (TAL)polypeptides and compositions thereof. In some embodiments, theengineered TAL polypeptides have been optimized to provide enhancedcatalytic activity and enhanced acid stability, while reducingsensitivity to proteolysis. The invention also provides methods forutilization of the compositions comprising the engineered TALpolypeptides for therapeutic and industrial purposes.

Abbreviations and Definitions:

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention pertains. Generally,the nomenclature used herein and the laboratory procedures of cellculture, molecular genetics, microbiology, organic chemistry, analyticalchemistry and nucleic acid chemistry described below are thosewell-known and commonly employed in the art. Such techniques arewell-known and described in numerous texts and reference works wellknown to those of skill in the art. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses. All patents, patent applications, articles and publicationsmentioned herein, both supra and infra, are hereby expresslyincorporated herein by reference.

Although any suitable methods and materials similar or equivalent tothose described herein find use in the practice of the presentinvention, some methods and materials are described herein. It is to beunderstood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context they are used by those of skill in the art.Accordingly, the terms defined immediately below are more fullydescribed by reference to the application as a whole. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by reference.

Also, as used herein, the singular “a”, “an,” and “the” include theplural references, unless the context clearly indicates otherwise.

Numeric ranges are inclusive of the numbers defining the range. Thus,every numerical range disclosed herein is intended to encompass everynarrower numerical range that falls within such broader numerical range,as if such narrower numerical ranges were all expressly written herein.It is also intended that every maximum (or minimum) numerical limitationdisclosed herein includes every lower (or higher) numerical limitation,as if such lower (or higher) numerical limitations were expresslywritten herein.

The term “about” means an acceptable error for a particular value. Insome instances “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of agiven value range. In some instances, “about” means within 1, 2, 3, or 4standard deviations of a given value. In some instances, “about”encompasses values that are within 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%,6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention which can be had byreference to the application as a whole. Accordingly, the terms definedimmediately below are more fully defined by reference to the applicationas a whole. Nonetheless, in order to facilitate understanding of theinvention, a number of terms are defined below.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

“EC” number refers to the Enzyme Nomenclature of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology (NC-IUBMB). The IUBMB biochemical classification is a numericalclassification system for enzymes based on the chemical reactions theycatalyze.

“ATCC” refers to the American Type Culture Collection whosebiorepository collection includes genes and strains.

“NCBI” refers to National Center for Biological Information and thesequence databases provided therein.

As used herein, the terms “tyrosine ammonia-lyase” “tyrosine ammonialyase,” “tyrosine ammonia lyase polypeptide” and “TAL” refer to a classof enzymes within the aromatic amino acid lyase family (EC 4.3.1.23, EC4.3.1.24 and EC4.3.1.25) which also includes histidine ammonia-lyase,and phenylalanine ammonia-lyase.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation).

“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes.“Amino acids” are referred to herein by either their commonly knownthree-letter symbols or by the one-letter symbols recommended byIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single letter codes. Theabbreviations used for the genetically encoded amino acids areconventional and are as follows: alanine (Ala or A), arginine (Arg orR), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C),glutamate (Glu or E), glutamine (Gln or Q), histidine (His or H),isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine(Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser orS), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y),and valine (Val or V). When the three-letter abbreviations are used,unless specifically preceded by an “L” or a “D” or clear from thecontext in which the abbreviation is used, the amino acid may be ineither the L- or D-configuration about α-carbon (C_(α)). For example,whereas “Ala” designates alanine without specifying the configurationabout the α-carbon, “D-Ala” and “L-Ala” designate D-alanine andL-alanine, respectively. When the one-letter abbreviations are used,upper case letters designate amino acids in the L-configuration aboutthe a-carbon and lower case letters designate amino acids in theD-configuration about the α-carbon. For example, “A” designatesL-alanine and “a” designates D-alanine. When polypeptide sequences arepresented as a string of one-letter or three-letter abbreviations (ormixtures thereof), the sequences are presented in the amino (N) tocarboxy (C) direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleosides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

The term “engineered,” “recombinant,” “non-naturally occurring,” and“variant,” when used with reference to a cell, a polynucleotide or apolypeptide refers to a material or a material corresponding to thenatural or native form of the material that has been modified in amanner that would not otherwise exist in nature or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques.

As used herein, “wild-type” and “naturally-occurring” refer to the formfound in nature. For example a wild-type polypeptide or polynucleotidesequence is a sequence present in an organism that can be isolated froma source in nature and which has not been intentionally modified byhuman manipulation.

“Deimmunized” as used herein, refers to the manipulation of a protein tocreate a variant that is not as immunogenic as the wild-type orreference protein. In some embodiments, the deimmunization is complete,in that the variant protein does not stimulate an immune response inpatients to whom the variant protein is administered. This response canbe measured by various methods including but not limited to, thepresence or abundance of anti-drug antibodies, the presence or abundanceof neutralizing antibodies, the presence of an anaphylactic response, orthe prevalence or intensity of cytokine release upon administration ofthe protein. In some embodiments, the variant protein is lessimmunogenic than the wild-type or reference protein. In someembodiments, deimmunization involves modifications to proteins (e.g.,epitopes) that are recognized by T-cell receptors. In some embodiments,the T-cell epitopes are removed from a wild-type or reference protein inorder to produce a deimmunized variant protein. In some embodiments, thedeimmunized protein shows lower levels of response in biochemical andcell-biological predictors of human immunological responses includingdendritic-cell T-cell activation assays, or human leukocyte antigen(HLA) peptide binding assays.

“Coding sequence” refers to that part of a nucleic acid (e.g., a gene)that encodes an amino acid sequence of a protein.

The term “percent (%) sequence identity” is used herein to refer tocomparisons among polynucleotides and polypeptides, and are determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage may be calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Alternatively, thepercentage may be calculated by determining the number of positions atwhich either the identical nucleic acid base or amino acid residueoccurs in both sequences or a nucleic acid base or amino acid residue isaligned with a gap to yield the number of matched positions, dividingthe number of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Those of skill in the art appreciatethat there are many established algorithms available to align twosequences. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(Smith and Waterman, Adv. Appl. Math., 2:482 [1981]), by the homologyalignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.Mol. Biol., 48:443 [1970), by the search for similarity method ofPearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444 [1988]), by computerized implementations of these algorithms(e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin SoftwarePackage), or by visual inspection, as known in the art. Examples ofalgorithms that are suitable for determining percent sequence identityand sequence similarity include, but are not limited to the BLAST andBLAST 2.0 algorithms, which are described by Altschul et al. (See,Altschul et al., J. Mol. Biol., 215: 403-410 [1990]; and Altschul etal., 1977, Nucleic Acids Res., 3389-3402 [1977], respectively). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as, theneighborhood word score threshold (See, Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915[1989]). Exemplary determination of sequence alignment and % sequenceidentity can employ the BESTFIT or GAP programs in the GCG WisconsinSoftware package (Accelrys, Madison Wis.), using default parametersprovided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, at least 100 residues in length or the fulllength of the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides or polypeptides over a “comparison window” to identifyand compare local regions of sequence similarity. In some embodiments, a“reference sequence” can be based on a primary amino acid sequence,where the reference sequence is a sequence that can have one or morechanges in the primary sequence.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredTAL, can be aligned to a reference sequence by introducing gaps tooptimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a differencein the amino acid residue at a position of a polypeptide sequencerelative to the amino acid residue at a corresponding position in areference sequence. The positions of amino acid differences generallyare referred to herein as “Xn,” where n refers to the correspondingposition in the reference sequence upon which the residue difference isbased. For example, a “residue difference at position X93 as compared toSEQ ID NO: 2” refers to a difference of the amino acid residue at thepolypeptide position corresponding to position 93 of SEQ ID NO: 2. Thus,if the reference polypeptide of SEQ ID NO: 2 has a leucine at position93, then a “residue difference at position X93 as compared to SEQ ID NO:2” has an amino acid substitution of any residue other than leucine atthe position of the polypeptide corresponding to position 93 of SEQ IDNO: 2. In most instances herein, the specific amino acid residuedifference at a position is indicated as “XnY” where “Xn” specified thecorresponding position as described above, and “Y” is the single letteridentifier of the amino acid found in the engineered polypeptide (i.e.,the different residue than in the reference polypeptide). In someinstances, the present disclosure also provides specific amino aciddifferences denoted by the conventional notation “AnB”, where A is thesingle letter identifier of the residue in the reference sequence, “n”is the number of the residue position in the reference sequence, and Bis the single letter identifier of the residue substitution in thesequence of the engineered polypeptide. In some instances, a polypeptideof the present disclosure can include one or more amino acid residuedifferences relative to a reference sequence, which is indicated by alist of the specified positions where residue differences are presentrelative to the reference sequence. In some embodiments, where more thanone amino acid can be used in a specific residue position of apolypeptide, the various amino acid residues that can be used areseparated by a “/” (e.g., X307H/X307P or X307H/P). The presentapplication includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid (e.g., alanine, valine, leucine, and isoleucine); an amino acidwith hydroxyl side chain is substituted with another amino acid with ahydroxyl side chain (e.g., serine and threonine); an amino acids havingaromatic side chains is substituted with another amino acid having anaromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, andhistidine); an amino acid with a basic side chain is substituted withanother amino acid with a basis side chain (e.g., lysine and arginine);an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain (e.g., aspartic acid or glutamicacid); and/or a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively.

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered transaminase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. Insertions can be inthe internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

A “functional fragment” or a “biologically active fragment” usedinterchangeably herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion(s) and/or internaldeletions, but where the remaining amino acid sequence is identical tothe corresponding positions in the sequence to which it is beingcompared (e.g., a full-length engineered TAL of the present invention)and that retains substantially all of the activity of the full-lengthpolypeptide.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it (e.g.,protein, lipids, and polynucleotides). The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The recombinant TAL polypeptides may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, therecombinant TAL polypeptides can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure TAL composition comprisesabout 60% or more, about 70% or more, about 80% or more, about 90% ormore, about 95% or more, and about 98% or more of all macromolecularspecies by mole or % weight present in the composition. In someembodiments, the object species is purified to essential homogeneity(i.e., contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species. In some embodiments, the isolated recombinantTAL polypeptides are substantially pure polypeptide compositions.

“Improved enzyme property” refers to an engineered TAL polypeptide thatexhibits an improvement in any enzyme property as compared to areference TAL polypeptide and/or as a wild-type PAL polypeptide (e.g.,the wild-type sequence of SEQ ID NO: 2) or another engineered TALpolypeptide. Improved properties include but are not limited to suchproperties as increased protein expression, increased thermoactivity,increased thermostability, increased pH activity, increased stability,increased enzymatic activity, increased substrate specificity oraffinity, increased specific activity, increased resistance to substrateor end-product inhibition, increased chemical stability, improvedchemoselectivity, improved solvent stability, increased tolerance toacidic pH, increased tolerance to proteolytic activity (i.e., reducedsensitivity to proteolysis), reduced aggregation, increased solubility,reduced immunogenicity, and altered temperature profile.

“Increased enzymatic activity” or “enhanced catalytic activity” refersto an improved property of the engineered TAL polypeptides, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of TAL) as compared to the reference TAL enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.1 fold the enzymatic activity ofthe corresponding wild-type enzyme, to as much as 2-fold, 5-fold,10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold,200-fold or more enzymatic activity than the naturally occurring TAL oranother engineered TAL from which the TAL polypeptides were derived.

In some embodiments, the engineered TAL polypeptides have a k_(cat) ofat least 0.1/sec, at least 0.2/sec, at least 0.3/sec, at least 0.5/sec,at least 1.0/sec and in some preferred embodiments greater than 1.0/sec.In some embodiments, the K_(m) is in the range of about 1 μm to about 5mM; in the range of about 5 μm to about 2 mM; in the range of about10 μmto about 2 mM; or in the range of about 10 μm to about 1 mM. In somespecific embodiments, the engineered TAL enzyme exhibits improvedenzymatic activity in the range of 1.5 to 10 fold, 1.5 to 25 fold, 1.5to 50 fold, 1.5 to 100 fold or greater than that of a reference TALenzyme (e.g., a wild-type TAL or any other reference TAL). TAL activitycan be measured by any suitable method known in the art (e.g., standardassays, such as monitoring changes in spectrophotometric properties ofreactants or products). In some embodiments, the amount of productsproduced can be measured by High-Performance Liquid Chromatography(HPLC) separation combined with UV absorbance or fluorescent detectiondirectly or following o-phthaldialdehyde (OPA) derivatization. In someembodiments, other methods are used, such as tracking the coumarateproduct (e.g., use UV absorbance to track its production at 290 nm or310 nm). In some other embodiments, the production of ammonia is assayedusing commercially available kits (e.g., the Megazyme rapid ammoniaassay kit [Megazyme International, Wicklow, Ireland]). Comparisons ofenzyme activities are made using a defined preparation of enzyme, adefined assay under a set condition, and one or more defined substrates,as further described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

The terms “thermally stable” and “thermostable” refer to enzymes of thepresent invention that retain a specified amount of enzymatic activity,primary, secondary, tertiary and quaternary structure after exposure toidentified temperatures over a given period of time under conditionsprevailing during the use of the enzyme, for example, when exposed toaltered temperatures. “Altered temperatures” include increased ordecreased temperatures. In some embodiments, the enzymes retain at leastabout 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99%enzymatic activity after exposure to altered temperatures over a giventime period, for example, at least about 60 minutes, about 120 minutes,about 180 minutes, about 240 minutes, about 300 minutes, etc.

The term “improved tolerance to acidic pH” means that a recombinant TALaccording to the invention will have increased stability (higherretained activity at about pH 7.0 after exposure to acidic pH for aspecified period of time (1 hour, up to 24 hours)) as compared to areference TAL or another enzyme.

“Physiological pH” as used herein means the pH range generally found ina subject's (e.g., human) small intestine. There normally is a pHgradient from the pyloric sphincter to the large intestine from about5.0 to 7.5.

The term “acidic pH” (e.g., used with reference to improved stability toacidic pH conditions or increased tolerance to acidic pH) means a pHrange of about 1.5 to 6.8.

The terms “proteolytic activity” and “proteolysis” used interchangeablyherein refer to the breakdown of proteins into smaller polypeptides oramino acids. The breakdown of proteins is generally the result ofhydrolysis of the peptide bond by protease (proteinase) enzymes.Protease enzymes include but are not limited to pepsin, trypsin,chymotrypsin, elastase, carboxypeptidase A and B, peptidases (e.g.,amino peptidase, dipeptidase and enteropeptidase).

The phrases “reducing sensitivity to proteolysis” and “reducingproteolytic sensitivity” used interchangeably herein mean that anengineered TAL polypeptide according to the invention will have a higherenzyme activity compared to a reference TAL and/or another enzyme in astandard assay after treatment with one or more proteases. Exemplaryassays are provided in the Examples.

“Aggregation” means clumping or precipitation of a TAL polypeptide.Aggregation can lead to inactivation, and/or increased immunogenicity ofthe enzyme. The term “reduced aggregation” means an engineered TALpolypeptide will be less prone to aggregation or to aggregate than areference TAL and/or another enzyme. Methods for determining Aggregationcan be determined by one of general skill in the art by using any numberof assays including but not limited to fluorescent microscopy withappropriate dyes (e.g., thioflavin T or Nile Red), dynamic lightscattering, flow cytometry with appropriate dyes (e.g. Bodipy),filtration and analysis by SDS-PAGE or Western blotting, fluorescentcorrelation spectroscopy, and electron microscopy. There arecommercially available kits to assess aggregation (e.g., the PROTEOSTAT®Protein Aggregation Assay kit [Enzo]).

“Conversion” refers to the enzymatic conversion (or biotransformation)of a substrate(s) to the corresponding product(s). “Percent conversion”refers to the percent of the substrate that is converted to the productwithin a period of time under specified conditions. Thus, the “enzymaticactivity” or “activity” of a TAL polypeptide can be expressed as“percent conversion” of the substrate to the product in a specificperiod of time.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is more efficiently expressed in theorganism of interest. Although the genetic code is degenerate in thatmost amino acids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theTAL enzymes may be codon optimized for optimal production from the hostorganism selected for expression.

“Control sequence” refers herein to include all components, which arenecessary or advantageous for the expression of a polynucleotide and/orpolypeptide of the present application. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter sequence, signalpeptide sequence, initiation sequence and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refers to those conditions in theenzymatic conversion reaction solution (e.g., ranges of enzyme loading,substrate loading, temperature, pH, buffers, co-solvents, etc.) underwhich a TAL polypeptide of the present application is capable ofconverting a substrate to the desired product compound, Exemplary“suitable reaction conditions” are provided in the present applicationand illustrated by the Examples. “Loading”, such as in “compoundloading” or “enzyme loading” refers to the concentration or amount of acomponent in a reaction mixture at the start of the reaction.“Substrate” in the context of an enzymatic conversion reaction processrefers to the compound or molecule acted on by the TAL polypeptide.“Product” in the context of an enzymatic conversion process refers tothe compound or molecule resulting from the action of the TALpolypeptide on a substrate.

As used herein the term “culturing” refers to the growing of apopulation of microbial cells under any suitable conditions (e.g., usinga liquid, gel or solid medium).

Recombinant polypeptides can be produced using any suitable methodsknown the art. Genes encoding the wild-type polypeptide of interest canbe cloned in vectors, such as plasmids, and expressed in desired hosts,such as E. coli, etc. Variants of recombinant polypeptides can begenerated by various methods known in the art. Indeed, there is a widevariety of different mutagenesis techniques well known to those skilledin the art. In addition, mutagenesis kits are also available from manycommercial molecular biology suppliers. Methods are available to makespecific substitutions at defined amino acids (site-directed), specificor random mutations in a localized region of the gene (regio-specific),or random mutagenesis over the entire gene (e.g., saturationmutagenesis). Numerous suitable methods are known to those in the art togenerate enzyme variants, including but not limited to site-directedmutagenesis of single-stranded DNA or double-stranded DNA using PCR,cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, andchemical saturation mutagenesis, or any other suitable method known inthe art. Non-limiting examples of methods used for DNA and proteinengineering are provided in the following patents: U.S. Pat. Nos.6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393;7,783,428; and 8,383,346. After the variants are produced, they can bescreened for any desired property (e.g., high or increased activity, orlow or reduced activity, increased thermal activity, increased thermalstability, and/or acidic pH stability, etc.). In some embodiments,“recombinant TAL polypeptides” (also referred to herein as “engineeredTAL polypeptides,” “variant TAL enzymes,” and “TAL variants”) find use.

As used herein, a “vector” is a DNA construct for introducing a DNAsequence into a cell. In some embodiments, the vector is an expressionvector that is operably linked to a suitable control sequence capable ofeffecting the expression in a suitable host of the polypeptide encodedin the DNA sequence. In some embodiments, an “expression vector” has apromoter sequence operably linked to the DNA sequence (e.g., transgene)to drive expression in a host cell, and in some embodiments, alsocomprises a transcription terminator sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation, andpost-translational modification. In some embodiments, the term alsoencompasses secretion of the polypeptide from a cell.

As used herein, the term “produces” refers to the production of proteinsand/or other compounds by cells. It is intended that the term encompassany step involved in the production of polypeptides including, but notlimited to, transcription, post-transcriptional modification,translation, and post-translational modification. In some embodiments,the term also encompasses secretion of the polypeptide from a cell.

As used herein, an amino acid or nucleotide sequence (e.g., a promotersequence, signal peptide, terminator sequence, etc.) is “heterologous”to another sequence with which it is operably linked if the twosequences are not associated in nature.

As used herein, the terms “host cell” and “host strain” refer tosuitable hosts for expression vectors comprising DNA provided herein(e.g., the polynucleotides encoding the TAL variants). In someembodiments, the host cells are prokaryotic or eukaryotic cells thathave been transformed or transfected with vectors constructed usingrecombinant DNA techniques as known in the art.

The term “analogue” means a polypeptide having more than 70% sequenceidentity but less than 100% sequence identity (e.g., more than 75%, 78%,80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity) with a reference polypeptide. In some embodiments,analogues means polypeptides that contain one or more non-naturallyoccurring amino acid residues including, but not limited, tohomoarginine, ornithine and norvaline, as well as naturally occurringamino acids. In some embodiments, analogues also include one or moreD-amino acid residues and non-peptide linkages between two or more aminoacid residues.

The term “therapeutic” refers to a compound administered to a subjectwho shows signs or symptoms of pathology having beneficial or desirablemedical effects.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject (e.g., human) comprising apharmaceutically effective amount of an engineered TAL polypeptideencompassed by the invention and an acceptable carrier.

The term “effective amount” means an amount sufficient to produce thedesired result. One of general skill in the art may determine what theeffective amount by using routine experimentation.

The terms “isolated” and “purified” are used to refer to a molecule(e.g., an isolated nucleic acid, polypeptide, etc.) or other componentthat is removed from at least one other component with which it isnaturally associated. The term “purified” does not require absolutepurity, rather it is intended as a relative definition.

The term “subject” encompasses mammals such as humans, non-humanprimates, livestock, companion animals, and laboratory animals (e.g.,rodents and lagamorphs). It is intended that the term encompass femalesas well as males.

As used herein, the term “patient” means any subject that is beingassessed for, treated for, or is experiencing disease.

The term “infant” refers to a child in the period of the first monthafter birth to approximately one (1) year of age. As used herein, theterm “newborn” refers to child in the period from birth to the 28^(th)day of life. The term “premature infant” refers to an infant born afterthe twentieth completed week of gestation, yet before full term,generally weighing ˜500 to ˜2499 grams at birth. A “very low birthweight infant” is an infant weighing less than 1500 g at birth.

As used herein, the term “child” refers to a person who has not attainedthe legal age for consent to treatment or research procedures. In someembodiments, the term refers to a person between the time of birth andadolescence.

As used herein, the term “adult” refers to a person who has attainedlegal age for the relevant jurisdiction (e.g., 18 years of age in theUnited States). In some embodiments, the term refers to any fully grown,mature organism. In some embodiments, the term “young adult” refers to aperson less than 18 years of age, but who has reached sexual maturity.

As used herein, “composition” and “formulation” encompass productscomprising at least one engineered TAL of the present invention,intended for any suitable use (e.g., pharmaceutical compositions,dietary/nutritional supplements, feed, etc.).

The terms “administration” and “administering” a composition meanproviding a composition of the present invention to a subject (e.g., toa person suffering from the effects of tyrosinemia or alkaptonuria).

The term “carrier” when used in reference to a pharmaceuticalcomposition means any of the standard pharmaceutical carrier, buffers,and excipients, such as stabilizers, preservatives, and adjuvants.

The term “pharmaceutically acceptable” means a material that can beadministered to a subject without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents in which it is contained and that possesses the desiredbiological activity.

As used herein, the term “excipient” refers to any pharmaceuticallyacceptable additive, carrier, diluent, adjuvant, or other ingredient,other than the active pharmaceutical ingredient (API; e.g., theengineered TAL polypeptides of the present invention). Excipients aretypically included for formulation and/or administration purposes.

The term “therapeutically effective amount” when used in reference tosymptoms of disease/condition refers to the amount and/or concentrationof a compound (e.g., engineered TAL polypeptides) that ameliorates,attenuates, or eliminates one or more symptom of a disease/condition orprevents or delays the onset of symptom(s).

The term “therapeutically effective amount” when used in reference to adisease/condition refers to the amount and/or concentration of acomposition (e.g., engineered TAL polypeptides) that ameliorates,attenuates, or eliminates the disease/condition. In some embodiments,the term is use in reference to the amount of a composition that elicitsthe biological (e.g., medical) response by a tissue, system, or animalsubject that is sought by the researcher, physician, veterinarian, orother clinician.

It is intended that the terms “treating,” “treat” and “treatment”encompass preventative (e.g., prophylactic), as well as palliativetreatment.

Engineered TAL Polypeptides:

The parent enzyme used to generate the engineered TAL polypeptides isselected from enzymes obtained from Stanieria cyanosphera (SEQ ID NO:2), Chroogloeocystis siderophila (SEQ ID NO: 4), Flavobacteriumjohnsoniae (SEQ ID NO: 6), and Rhodotorula glutinis (SEQ ID NO: 8). Insome embodiments, the engineered TAL polypeptides are derived bydirected evolution from S. cyanosphera. Furthermore, when a particularTAL variant (engineered TAL polypeptide) is referred to by reference tomodification of particular amino acids residues in the sequence of awild-type TAL, wild-type PAL, wild-type HAL or another TAL, it is to beunderstood that variants of another TAL modified in the equivalentposition(s) (as determined from the optional amino acid sequencealignment between the respective amino acid sequences) are encompassedherein. In some embodiments the engineered TAL polypeptide will comprisethe conserved active site Ala164-Ser165-Gly166 and comprise at least 70%(at least 75%, at least 80%, at least 85%, at least 90%, at least 93%,at least 95%, at least 97%) sequence identity to SEQ ID NO: 2. In someembodiments the engineered TAL polypeptides comprise not only TALactivity but also may be active on phenylalanine and/or histidinesubstrates. In some embodiments, TAL variants are developed thatcomprise at least one mutation in at least one key residue (See e.g., WO2008/069958; US Appln. Ser. No. 2009/011140; and Watts et al. Chem.Biol., 13:1317-26 [2006]).

In some embodiments, engineered TAL polypeptides are produced bycultivating a microorganism comprising at least one polynucleotidesequence encoding at least one engineered TAL polypeptide underconditions which are conducive for producing the engineered TALpolypeptide(s). In some embodiments, the engineered TAL polypeptide isrecovered from the resulting culture medium and/or cells.

The present invention provides exemplary engineered TAL polypeptideshaving TAL activity. The Examples provide Tables showing sequencestructural information correlating specific amino acid sequence featureswith the functional activity of the engineered TAL polypeptides. Thisstructure-function correlation information is provided in the form ofspecific amino acid residues differences relative to a referenceengineered polypeptide, as indicated in the Examples. The Examplesfurther provide experimentally determined activity data for theexemplary engineered TAL polypeptides.

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at leastabout 85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156; b) an amino acid residue difference as compared to SEQ ID NO: 2,14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156, at one or more amino acid positions; and c) which exhibitsan improved property selected from i) enhanced catalytic activity, ii)reduced proteolytic sensitivity, iii) increased tolerance to acidic pH,iv) reduced aggregation or a combination of any of i), ii), iii) or iv),as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454,1578, 1660, 1844, 2030, 2114, and/or 2156, and/or another referencesequence.

In some embodiments the engineered TAL which exhibits an improvedproperty has at least about 85%, at least about 88%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or at about 100% amino acidsequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790,1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, and an amino acidresidue difference as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, at one ormore amino acid positions (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO: 2, ora sequence having at least 85%, at least 88%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or greater amino acidsequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790,1454, 1578, 1660, 1844, 2030, 2114, and/or 2156). In some embodiment theresidue difference as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, at one ormore positions will include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore conservative amino acid substitutions. In some embodiments theengineered TAL which exhibits an improved property has at least 85%, atleast 88%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% amino acid sequence identity with SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156.

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156; b)an amino acid residue difference as compared to SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156, at one or more amino acid positions; and c) which exhibits animproved property selected from i) enhanced catalytic activity, ii)reduced proteolytic sensitivity, iii) increased tolerance to acidic pH,iv) reduced aggregation or a combination of any of i), ii), iii) or iv),as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454,1578, 1660, 1844, 2030, 2114, and/or 2156, and/or another referencesequence.

In some embodiments, the engineered TAL polypeptide is a polypeptidelisted in Table 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 9-2, 10-1,10-2, 11-1, 12-1, 13-1, 14-1, 15-1, 16-1, and/or 17-1.

In some embodiments, the engineered TAL polypeptide comprises afunctional fragment of an engineered TAL polypeptide encompassed by theinvention. Functional fragments have at least 95%, 96%, 97%, 98%, or 99%of the activity of the engineered TAL polypeptide from which is wasderived (i.e., the parent engineered TAL). A functional fragmentcomprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and even99% of the parent sequence of the engineered TAL. In some embodimentsthe functional fragment is truncated by less than 5, less than 10, lessthan 15, less than 10, less than 25, less than 30, less than 35, lessthan 40, less than 45, and less than 50 amino acids.

Variants with Reduced Sensitivity to Proteolysis:

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156; b)an amino acid residue difference as compared to SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156, at one or more amino acid positions; and c) which exhibits reducedsensitivity to proteolysis as compared to SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156,and/or another reference sequence.

In some embodiments the engineered TAL which exhibits reducedsensitivity to proteolysis has at least 85%, at least 88%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or greater aminoacid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604, 736,790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, and an amino acidresidue difference as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, at one ormore amino acid positions (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO: : 2,14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156, or a sequence having at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or greateramino acid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156).

In some embodiments, the proteolytic sensitivity of the engineered TALpolypeptide will be reduced by at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% and at least 85% compared to thewild-type Stanieria cyanosphaera enzyme of SEQ ID NO:2, and/or at leastone reference TAL polypeptide under essentially the same conditions. Theproteolytic sensitivity can be measured using any suitable assay system,including, but not limited to the assays described in the Examples.

In some embodiments, the engineered TAL polypeptide having reducedsensitivity to proteolysis has reduced sensitivity to a compositioncomprising one or more proteases such as but not limited to pepsin,trypsin, chymotrypsin, carboxypeptidase A and B, peptidases (e.g., aminopeptidase, dipeptidase and enteropeptidase) when both the reference TALand the engineered TAL having the reduced sensitivity are compared andexposed to essentially the same amount and kind of protease underessentially the same conditions.

In some embodiments, the engineered TAL polypeptide having reducedsensitivity to proteolysis has enzyme activity that is about 1.0 fold,2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold,150-fold, 200-fold or more of the enzymatic activity of the referenceTAL. In some embodiments, the engineered polypeptides will have moreenzyme activity (as compared to a reference TAL) when activity ismeasured at a pH range of 4.5 to 7.5; when activity is measured at a pHrange of 4.5 to 6.5; when activity is measured at a pH range of 5.0 to7.5, when activity is measured at a pH range of 5.0 to 6.5; whenactivity is measured at a pH range of 5.5 to 7.5 and also when activityis measured at a pH range of 5.5 to 6.5. In other embodiments, theengineered TAL polypeptides will have a Km in the range of 1 μM to 5 mM.

Variants with Increased Tolerance to Acidic pH:

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, ora fragment thereof; b) an amino acid residue difference as compared toSEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844,2030, 2114, and/or 2156, at one or more amino acid positions; and c)which exhibits increased tolerance to acidic pH as compared to SEQ IDNO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030,2114, and/or 2156, and/or another reference sequence.

In some embodiments, the engineered TAL that exhibits increasedtolerance to acidic pH has at least about 85%, at least about 88%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, or greateramino acid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, and an aminoacid residue difference as compared to SEQ ID NO: 2, 14, 86, 334, 388,604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, at oneor more amino acid positions (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO:2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156, or a sequence having at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or greateramino acid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156.

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, ora fragment thereof; b) an amino acid residue difference as compared toSEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844,2030, 2114, and/or 2156, at one or more amino acid positions; and c)which exhibits increased tolerance to acidic pH as compared to SEQ IDNO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030,2114, and/or 2156, and/or another reference sequence.

In some embodiments, the engineered TAL that exhibits increasedtolerance to acidic pH has at least 85%, at least 88%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%,or greater aminoacid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604, 736,790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, and an amino acidresidue difference as compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, at one ormore amino acid positions (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 14, 15, 20 or more amino acid positions compared to SEQ ID NO: 2,14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156, or a sequence having at least 85%, at least 88%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or greateramino acid sequence identity with SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156.

In some embodiments, when all other assay conditions are essentially thesame, the engineered TAL polypeptide has increased tolerance to acidicpH as compared to a reference TAL polypeptide. The engineered peptidehas an increased tolerance at a pH range between 1.5 to 6.5, and between1.5 and 5.0, and between 2.0 to 5.5, and between 3.0 and 6.8; between3.0 and 5.5; between 4.0 and 6.5; between 4.0 and 4.5; between 4.5 andbetween 5.0; between 4.5 and 5.5, between 4.5 and 6.0; between 4.5 and6.5; between 5.0 and 6.5; between 5.0 and 6.0; between 5.0 and 5.5;between 5.5 and 6.0; between 6.0 and 6.5 and between 6.5 and 7.0. Insome embodiments the increased tolerance to acidic pH will be exhibitedat a pH of about 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5.

In some embodiments, the engineered TAL polypeptide having increasedtolerance to acidic pH exhibits greater TAL activity as compared to areference TAL when measured by any standard assay, including, but notlimited to the assays described in the Examples.

Variants with Improved Activity:

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, ora fragment thereof; b) an amino acid residue difference as compared toSEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844,2030, 2114, and/or 2156, at one or more amino acid positions; and c)which exhibits improved activity, as compared to SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156, and/or another reference sequence.

In some embodiments, the engineered TAL that exhibits improved activityhas at least about 85%, at least about 88%, at least about 90%, at leastabout 91%, at least about 92%, at least about 93%, at least about 94%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or greater amino acid sequence identitywith SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660,1844, 2030, 2114, and/or 2156, and an amino acid residue difference ascompared to SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578,1660, 1844, 2030, 2114, and/or 2156, at one or more amino acid positions(e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or moreamino acid positions compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, or a sequencehaving at least about 85%, at least about 88%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or greater amino acid sequenceidentity with SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578,1660, 1844, 2030, 2114, and/or 2156.

In some embodiments, the engineered TAL polypeptides of the inventionhaving TAL activity comprise a) an amino acid sequence having at least85% sequence identity to reference sequence SEQ ID NO: 2, 14, 86, 334,388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, ora fragment thereof; b) an amino acid residue difference as compared toSEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844,2030, 2114, and/or 2156, at one or more amino acid positions; and c)which exhibits improved activity, as compared to SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156, and/or another reference sequence.

In some embodiments, the engineered TAL that exhibits improved activityhas at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660,1844, 2030, 2114, and/or 2156, and an amino acid residue difference ascompared to SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578,1660, 1844, 2030, 2114, and/or 2156, at one or more amino acid positions(e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or moreamino acid positions compared to SEQ ID NO: 2, 14, 86, 334, 388, 604,736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, or a sequencehaving at least 85%, at least 88%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or greater amino acid sequence identitywith SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660,1844, 2030, 2114, and/or 2156.

In some embodiments, when all other assay conditions are essentially thesame, the engineered TAL polypeptide has improved activity as comparedto a reference TAL polypeptide. In some embodiments this activity can bemeasured under conditions that monitor enzymatic activity at saturatinglevels of tyrosine, thus assessing the maximum activity of the enzyme(k_(cat)). In other embodiments this activity can be measured undersubstrate concentrations resulting in one-half, one-fifth, one-tenth orless of maximal activity. Under either method of analysis, theengineered polypeptide has improved activity levels about 1.0 fold,1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold,100-fold, or more of the enzymatic activity of the reference TAL In someembodiments, the engineered TAL polypeptide having improved activity ascompared to a reference TAL when measured by any standard assay,including, but not limited to the assays described in the Examples.

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptides (e.g., SEQ ID NOS:10-2290; in particular SEQ ID NOS: 2, 14, 86, 334, 388, 604, 736, 790,1454, 1578, 1660, 1844, 2030, 2114, and/or 2156) can be used as thestarting amino acid sequence for synthesizing other engineered TALpolypeptides, for example by subsequent rounds of evolution by addingnew combinations of various amino acid differences from otherpolypeptides and other residue positions described herein. Furtherimprovements may be generated by including amino acid differences atresidue positions that had been maintained as unchanged throughoutearlier rounds of evolution.

Polynucleotides Encoding Engineered Polypeptides, Expression Vectors andHost Cells:

The present invention provides polynucleotides encoding the engineeredTAL polypeptides described herein. In some embodiments, thepolynucleotides are operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered TAL polypeptides can be introduced into appropriate hostcells to express the corresponding TAL polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the engineered TAL polypeptide. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the variants provided inTable 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 9-2, 10-1, 10-2,11-1, 12-1, 13-1, 14-1, 15-1, 16-1, and/or 17-1, as well as SEQ ID NOS:2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used for expression in bacteria.Consequently, codon optimized polynucleotides encoding the engineeredTAL polypeptides contain preferred codons at about 40%, 50%, 60%, 70%,80%, or greater than 90% of codon positions of the full length codingregion.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having TAL activity with the properties disclosedherein, wherein the polypeptide comprises an amino acid sequence havingat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identity to a reference sequence selectedfrom SEQ ID NOS: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660,1844, 2030, 2114, and/or 2156, or the amino acid sequence of any variantas disclosed in Tables 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 9-2,10-1, 10-2, 11-1, 12-1, 13-1, 14-1, 15-1, 16-1, and/or 17-1, and one ormore residue differences as compared to the reference polypeptide of SEQID NOS: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844,2030, 2114, and/or 2156, or the amino acid sequence of any variant asdisclosed in Table 1-1, 2-1, 3-1, 4-1, 5-1, 6-1, 7-1, 8-1, 9-1, 9-2,10-1, 10-2, 11-1, 12-1, 13-1, 14-1, 15-1, 16-1, and/or 17-1 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions). In someembodiments, the reference sequence is selected from SEQ ID NOS: 2, 14,86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156.

In some embodiments, the polynucleotide encoding the engineered TALpolypeptides comprises a polynucleotide sequence selected from apolynucleotide sequence encoding the SEQ ID NOS: 3, 13, 85, 333, 387,603, 735, 789, 1453, 1577, 1659, 1843, 2029, 2113, and/or 2155. In someembodiments, the polynucleotide encoding an engineered TAL polypeptidehas at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotideresidue identity to SEQ ID NOS: 3, 13, 85, 333, 387, 603, 735, 789,1453, 1577, 1659, 1843, 2029, 2113, and/or 2155. In some embodiments,the polynucleotides are capable of hybridizing under highly stringentconditions to a reference polynucleotide sequence selected from SEQ IDNOS: 3, 13, 85, 333, 387, 603, 735, 789, 1453, 1577, 1659, 1843, 2029,2113, and/or 2155, or a complement thereof, or a polynucleotide sequenceencoding any of the variant TAL polypeptides provided herein.

In some embodiments, an isolated polynucleotide encoding any of theengineered TAL polypeptides provided herein is manipulated in a varietyof ways to provide for expression of the polypeptide. In someembodiments, the polynucleotides encoding the polypeptides are providedas expression vectors where one or more control sequences is present toregulate the expression of the polynucleotides and/or polypeptides.Manipulation of the isolated polynucleotide prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotides and nucleic acidsequences utilizing recombinant DNA methods are well known in the art.

In some embodiments, the control sequences include among othersequences, promoters, leader sequences, polyadenylation sequences,propeptide sequences, signal peptide sequences, and transcriptionterminators. As known in the art, suitable promoters can be selectedbased on the host cells used. For bacterial host cells, suitablepromoters for directing transcription of the nucleic acid constructs ofthe present application, include, but are not limited to the promotersobtained from the E. coli lac operon, Streptomyces coelicolor agarasegene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Seee.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731[1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc.Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promoters forfilamentous fungal host cells, include promoters obtained from the genesfor Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (ahybrid of the promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase), andmutant, truncated, and hybrid promoters thereof. Exemplary yeast cellpromoters can be from the genes can be from the genes for Saccharomycescerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase(GALL), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are known in the art (See e.g., Romanoset al., Yeast 8:423-488 [1992]).

In some embodiments, the control sequence is a suitable transcriptionterminator sequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice finds use inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are known in the art (See e.g., Romanos et al., supra).

In some embodiments, the control sequence is a suitable leader sequence,a non-translated region of an mRNA that is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used.Exemplary leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cellsinclude, but are not limited to those obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells include, but are not limited to those fromthe genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease, and Aspergillus nigeralpha-glucosidase. Useful polyadenylation sequences for yeast host cellsare also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol.,15:5983-5990 [1995]).

In some embodiments, the control sequence is a signal peptide codingregion that codes for an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. Any signal peptide codingregion that directs the expressed polypeptide into the secretory pathwayof a host cell of choice finds use for expression of the engineered TALpolypeptides provided herein. Effective signal peptide coding regionsfor bacterial host cells include, but are not limited to the signalpeptide coding regions obtained from the genes for Bacillus NClB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57:109-137 [1993]).Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase. Useful signal peptides for yeast host cells include,but are not limited to those from the genes for Saccharomyces cerevisiaealpha-factor and Saccharomyces cerevisiae invertase.

In some embodiments, the control sequence is a propeptide coding regionthat codes for an amino acid sequence positioned at the amino terminusof a polypeptide. The resultant polypeptide is referred to as a“proenzyme,” “propolypeptide,” or “zymogen,” in some cases). Apropolypeptide can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region includes, but is notlimited to the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836). Where both signal peptideand propeptide regions are present at the amino terminus of apolypeptide, the propeptide region is positioned next to the aminoterminus of a polypeptide and the signal peptide region is positionednext to the amino terminus of the propeptide region.

In some embodiments, regulatory sequences are also utilized. Thesesequences facilitate the regulation of the expression of the polypeptiderelative to the growth of the host cell. Examples of regulatory systemsare those which cause the expression of the gene to be turned on or offin response to a chemical or physical stimulus, including the presenceof a regulatory compound. In prokaryotic host cells, suitable regulatorysequences include, but are not limited to the lac, tac, and trp operatorsystems. In yeast host cells, suitable regulatory systems include, butare not limited to the ADH2 system or GAL1 system. In filamentous fungi,suitable regulatory sequences include, but are not limited to the TAKAalpha-amylase promoter, Aspergillus niger glucoamylase promoter, andAspergillus oryzae glucoamylase promoter.

In another aspect, the present invention also provides a recombinantexpression vector comprising a polynucleotide encoding an engineered TALpolypeptide, and one or more expression regulating regions such as apromoter and a terminator, a replication origin, etc., depending on thetype of hosts into which they are to be introduced. in some embodiments,the various nucleic acid and control sequences described above arejoined together to produce a recombinant expression vector whichincludes one or more convenient restriction sites to allow for insertionor substitution of the nucleic acid sequence encoding the variant TALpolypeptide at such sites. Alternatively, the polynucleotide sequence(s)of the present invention are expressed by inserting the polynucleotidesequence or a nucleic acid construct comprising the polynucleotidesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), that can be conveniently subjected to recombinant DNA proceduresand can result in the expression of the variant TAL polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extra-chromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extra-chromosomal element, aminichromosome, or an artificial chromosome). The vector may contain anymeans for assuring self-replication. In some alternative embodiments,the vector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon may be used.

In some embodiments, the expression vector preferably contains one ormore selectable markers, which permit easy selection of transformedcells. A “selectable marker” is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markersinclude, but are not limited to the dal genes from Bacillus subtilis orBacillus licheniformis, or markers, which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Suitable markers for yeast host cells include, but are notlimited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferases),bar (phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof. Inanother aspect, the present invention provides a host cell comprising apolynucleotide encoding at least one engineered TAL polypeptide of thepresent application, the polynucleotide being operatively linked to oneor more control sequences for expression of the engineered TAL enzyme(s)in the host cell. Host cells for use in expressing the polypeptidesencoded by the expression vectors of the present invention are wellknown in the art and include but are not limited to, bacterial cells,such as E. coli, Vibrio fluvialis, Streptomyces and Salmonellatyphimurium cells; fungal cells, such as yeast cells (e.g.,Saccharomyces cerevisiae and Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; andplant cells. Exemplary host cells are Escherichia coli strains (such asW3110 (ΔfhuA) and BL21).

Accordingly, in another aspect, the present invention provides methodsfor producing the engineered TAL polypeptides, where the methodscomprise culturing a host cell capable of expressing a polynucleotideencoding the engineered TAL polypeptide under conditions suitable forexpression of the polypeptide. In some embodiments, the methods furthercomprise the steps of isolating and/or purifying the TAL polypeptides,as described herein.

Appropriate culture media and growth conditions for the above-describedhost cells are well known in the art. Polynucleotides for expression ofthe TAL polypeptides may be introduced into cells by various methodsknown in the art. Techniques include, among others, electroporation,biolistic particle bombardment, liposome mediated transfection, calciumchloride transfection, and protoplast fusion.

The engineered TAL with the properties disclosed herein can be obtainedby subjecting the polynucleotide encoding the naturally occurring orengineered TAL polypeptide to mutagenesis and/or directed evolutionmethods known in the art, and as described herein. An exemplary directedevolution technique is mutagenesis and/or DNA shuffling (See e.g.,Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 [1994]; WO 95/22625;WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746). Other directed evolution procedures that can beused include, among others, staggered extension process (StEP), in vitrorecombination (See e.g., Zhao et al., Nat. Biotechnol., 16:258-261[1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Methods Appl.,3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al.,Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]).

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201,6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344,6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160,6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742,6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246,6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855,6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224,6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011,6,484,105, 6,489,146, 6,500,617, 6,500,639, 6,506,602, 6,506,603,6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098,6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514,6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, 6,902,922,6,917,882, 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515,7,105,297, 7,148,054, 7,220,566, 7,288,375, 7,384,387, 7,421,347,7,430,477, 7,462,469, 7,534,564, 7,620,500, 7,620,502, 7,629,170,7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428,7,795,030, 7,853,410, 7,868,138, 7,783,428, 7,873,477, 7,873,499,7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674,8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681,8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871,9,593,326, 9,684,771, 9,665,694; and WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; WO 01/75767; WO 2009/152336, WO2013/138339, WO 2015/048572, and WO 2015/048573; and all related US andnon-US counterparts of these listed patents and applications; Ling etal., Anal. Biochem., 254:157-78 [1997]; Dale et al., Meth. Mol. Biol.,57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botsteinet al., Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7[1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene,34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290[1999]; Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameriet al., Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; and Stemmer, Proc. Nat.Acad. Sci. USA, 91:10747-10751 [1994]; all of which are incorporatedherein by reference).

In some embodiments, the enzyme clones obtained following mutagenesistreatment are screened by subjecting the enzymes to a definedtemperature (or other assay conditions) and measuring the amount ofenzyme activity remaining after heat treatments or other assayconditions. Clones containing a polynucleotide encoding a TALpolypeptide are then isolated from the gene, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using any suitable method known in the art (e.g., standardbiochemistry techniques, such as HPLC analysis).

For engineered polypeptides of known sequence, the polynucleotidesencoding the enzyme can be prepared by standard solid-phase methods,according to known synthetic methods. In some embodiments, fragments ofup to about 100 bases can be individually synthesized, then joined(e.g., by enzymatic or chemical litigation methods, or polymerasemediated methods) to form any desired continuous sequence. For example,polynucleotides and oligonucleotides disclosed herein can be prepared bychemical synthesis using the classical phosphoramidite method (See e.g.,Beaucage et al., Tetra. Lett., 22:1859-69 [1981]; and Matthes et al.,EMBO J., 3:801-05 [1984]), as it is typically practiced in automatedsynthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors.

Accordingly, in some embodiments, a method for preparing the engineeredTAL polypeptide can comprise: (a) synthesizing a polynucleotide encodinga polypeptide comprising an amino acid sequence selected from the aminoacid sequence of any variant provided in Table 1-1, 2-1, 3-1, 4-1, 5-1,6-1, 7-1, 8-1, 9-1, 9-2, 10-1, 10-2, 11-1, 12-1, 13-1, 14-1, 15-1, 16-1,and/or 17-1, as well as SEQ ID NOS: 2, 14, 86, 334, 388, 604, 736, 790,1454, 1578, 1660, 1844, 2030, 2114, and/or 2156, and (b) expressing theTAL polypeptide encoded by the polynucleotide. In some embodiments ofthe method, the amino acid sequence encoded by the polynucleotide canoptionally have one or several (e.g., up to 3, 4, 5, or up to 10) aminoacid residue deletions, insertions and/or substitutions. In someembodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25,1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residuedeletions, insertions and/or substitutions. In some embodiments, thesubstitutions can be conservative or non-conservative substitutions.

The expressed engineered TAL polypeptide can be measured for any desiredimproved property (e.g., activity, selectivity, stability, acidtolerance, protease sensitivity, etc.), using any suitable assay knownin the art, including but not limited to the assays and conditionsdescribed herein.

In some embodiments, any of the engineered TAL polypeptides expressed ina host cell are recovered from the cells and/or the culture medium usingany one or more of the well-known techniques for protein purification,including, among others, lysozyme treatment, sonication, filtration,salting-out, ultra-centrifugation, and chromatography.

Chromatographic techniques for isolation of the TAL polypeptidesinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel electrophoresis, and affinitychromatography. Conditions for purifying a particular enzyme depends, inpart, on factors such as net charge, hydrophobicity, hydrophilicity,molecular weight, molecular shape, etc., and will be apparent to thosehaving skill in the art. In some embodiments, affinity techniques may beused to isolate the improved variant TAL enzymes. In some embodimentsutilizing affinity chromatography purification, any antibody whichspecifically binds the variant TAL polypeptide finds use. For theproduction of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc., are immunized by injection with aTAL polypeptide (e.g., a TAL variant), or a fragment thereof. In someembodiments, the TAL polypeptide or fragment is attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group.

In some embodiments, the engineered TAL polypeptide is produced in ahost cell by a method comprising culturing a host cell (e.g., an E. colistrain) comprising a polynucleotide sequence encoding an engineered TALpolypeptide as described herein under conditions conducive to theproduction of the engineered TAL polypeptide and recovering theengineered TAL polypeptide from the cells and/or culture medium.

In some preferred embodiments, the invention encompasses a method ofproducing an engineered TAL polypeptide comprising culturing arecombinant bacterial cell comprising a polynucleotide sequence encodingan engineered TAL polypeptide having at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to reference sequences SEQ ID NOS:2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114,and/or 2156, and one or more amino acid residue differences as comparedto SEQ ID NO: 2, 14, 86, 334, 388, 604, 736, 790, 1454, 1578, 1660,1844, 2030, 2114, and/or 2156, and/or combinations thereof when aligned,under suitable culture conditions to allow the production of theengineered TAL polypeptide and optionally recovering the engineered TALpolypeptide from the culture and/or cultured bacterial cells.

In some embodiments, once the engineered TAL polypeptides are recoveredfrom the recombinant host cells or cell culture and they are furtherpurified by any suitable method(s) known in the art. In some additionalembodiments, the purified TAL polypeptides are combined with otheringredients and compounds to provide compositions and formulationscomprising the engineered TAL polypeptide as appropriate for differentapplications and uses (e.g., pharmaceutical compositions).

Compositions:

Pharmaceutical Compositions

The present invention provides engineered TAL polypeptides suitable foruse in pharmaceutical and other compositions, such asdietary/nutritional supplements.

Depending on the mode of administration, the compositions comprising atherapeutically effective amount of an engineered TAL according to thepresent invention are in the form of a solid, semi-solid, gel, orliquid. In some embodiments, the compositions include otherpharmaceutically acceptable components such as diluents, buffers,excipients, salts, emulsifiers, preservatives, stabilizers, fillers, andother ingredients. Details on techniques for formulation andadministration are well known in the art and described in theliterature.

In some embodiments, the engineered TAL polypeptides are formulated foruse in oral pharmaceutical compositions. Any suitable format for use indelivering the engineered TAL polypeptides find use in the presentinvention, including but not limited to pills, tablets, gel tabs,capsules, lozenges, dragees, powders, soft gels, sol-gels, gels,emulsions, implants, patches, sprays, ointments, liniments, creams,pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks,suspensions (including but not limited to oil-based suspensions, oil-inwater emulsions, etc.), slurries, syrups, controlled releaseformulations, suppositories, etc. In some embodiments, the engineeredTAL polypeptides are provided in a format suitable for injection (i.e.,in an injectable formulation). In some embodiments, the engineered TALpolypeptides are provided in biocompatible matrices such as sol-gels,including silica-based (e.g., oxysilane) sol-gels. In some embodiments,the engineered TAL polypeptides are encapsulated. In some alternativeembodiments, the engineered TAL polypeptides are encapsulated innanostructures (e.g., nanotubes, nanotubules, nanocapsules, ormicrocapsules, microspheres, liposomes, etc.). Indeed, it is notintended that the present invention be limited to any particulardelivery formulation and/or means of delivery. It is intended that theengineered TAL polypeptides be administered by any suitable means knownin the art, including but not limited to parenteral, oral, topical,transdermal, intranasal, intraocular, intrathecal, via implants, etc.

In some embodiments, the engineered TAL polypeptides are chemicallymodified by glycosylation, pegylation (i.e., modified with polyethyleneglycol [PEG] or activated PEG, etc.) or other compounds (See e.g.,Ikeda, Amino Acids 29:283-287 [2005]; U.S. Pat. Nos. 7,531,341,7,534,595, 7,560,263, and 7,53,653; US Pat. Appln. Publ. Nos.2013/0039898, 2012/0177722, etc.). Indeed, it is not intended that thepresent invention be limited to any particular delivery method and/ormechanism.

In some additional embodiments, the engineered TAL polypeptides areprovided in formulations comprising matrix-stabilized enzyme crystals.In some embodiments, the formulation comprises a cross-linkedcrystalline engineered TAL enzyme and a polymer with a reactive moietythat adheres to the enzyme crystals. The present invention also providesengineered TAL polypeptides in polymers.

In some embodiments, compositions comprising the engineered TALpolypeptides of the present invention include one or more commonly usedcarrier compounds, including but not limited to sugars (e.g., lactose,sucrose, mannitol, and/or sorbitol), starches (e.g., corn, wheat, rice,potato, or other plant starch), cellulose (e.g., methyl cellulose,hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums(e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g,. gelatin,collagen, etc.). Additional components in oral formulations may includecoloring and or sweetening agents (e.g., glucose, sucrose, and mannitol)and lubricating agents (e.g., magnesium stearate), as well as entericcoatings (e.g., methacrylate polymers, hydroxyl propyl methyl cellulosephthalate, and/or any other suitable enteric coating known in the art).In some embodiments, disintegrating or solubilizing agents are included(e.g., cross-linked polyvinyl pyrrolidone, agar, alginic acid or saltsthereof, such as sodium alginate). In some embodiments, the engineeredTAL polypeptide are combined with various additional components,including but not limited to preservatives, suspending agents,thickening agents, wetting agents, alcohols, fatty acids, and/oremulsifiers, particularly in liquid formulations. In some embodiments,the engineered TAL polypeptides are administered to subjects incombination with other compounds used in the treatment of tyrosinemiaand/or alkaptonuria, including but not limited to NTBC, nitisinone,antacids (e.g., omeprazole, esomeprazole and other prazoles), as well asany other suitable compounds. In some additional embodiments, thepharmaceutical composition is suitable for parenteral injection into ahuman. In yet some additional embodiments, the pharmaceuticalcomposition is coadministered with a phenylalanine ammonia lyase. Insome further embodiments, the pharmaceutical composition comprisesphenylalanine ammonia lyase. In some embodiments, the pharmaceuticalcomposition comprises a pill, tablet, capsule, or gelcap that furthercomprises an enteric coating. In yet some additional embodiments, thepharmaceutical composition is coadministered with both nitisinone and aphenylalanine ammonia lyase. In some further embodiments, thepharmaceutical composition comprises both nitisinone and phenylalanineammonia lyase.

In some embodiments, the present invention provides engineered TALpolypeptides suitable for use in decreasing the concentration oftyrosine in fluids such as blood, cerebrospinal fluid, etc. The dosagesof engineered TAL polypeptide(s) administered to an animal depend uponthe condition or disease, the general condition of the animal, and otherfactors known to those in the art. In some embodiments, the compositionsare intended for single or multiple administrations to an animal. Insome embodiments, it is contemplated that the concentration ofengineered TAL polypeptide(s) in the composition(s) administered to ananimal (e.g., a human with tyrosinemia or alkaptonuria) is sufficient toeffectively treat, ameliorate and/or prevent the symptoms of disease(e.g., tyrosinemia or alkaptonuria and/or tyrosinemia oralkaptonuria-related conditions, diseases and/or symptoms), In someembodiments, the engineered TAL polypeptides are administered incombination with other pharmaceutical and/or dietary compositions.

Industrial Compositions

It is contemplated that the engineered TAL polypeptides of the presentinvention will find use in industrial compositions. In some embodiments,the engineered TAL polypeptides find use in the production of chemicals(e.g., coumaric acid). In some embodiments, the engineered TALpolypeptides are formulated for use in the food and/or feed industries.In some embodiments, the engineered TAL polypeptides are formulated ingranulated or pelleted products which are mixed with animal feedcomponents such as additional enzymes (for example, cellulases,laccases, and amylases).

In some alternative embodiments, the engineered TAL polypeptides areused in liquid animal feed compositions (e.g., aqueous or oil basedslurries). Thus, in some embodiments, the engineered TAL variants of thepresent invention are sufficiently thermotolerant and thermostable towithstand the treatment used to produce pellets and other processedfeed/foods. In some further embodiments, the engineered TAL variants areused to produce tyrosine and/or tyrosine derivatives.

The engineered TAL polypeptides provided herein also find use inagricultural applications. Indeed, it is contemplated that modulation ofTAL activity by using recombinant polypeptides having TAL activity willlead to effective herbicides.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following non-limiting examples. Theexamples are provided for illustrative purposes only and are notintended to limit the scope of the present invention in any way.

Experimental

The following Examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and l (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); CDS (coding sequence); DNA (deoxyribonucleicacid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratoryE. coli strain, available from the Coli Genetic Stock Center [CGSC], NewHaven, Conn.); LB (Luria-Burtani); TB (terrific broth); HPLC (highpressure liquid chromatography); SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis); PES (polyethersulfone); CFSE(carboxyfluorescein succinimidyl ester); IPTG (isopropylβ-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH(nicotinamide adenine dinucleotide phosphate); FIOPC (fold improvementsover positive control); PHE and Phe (phenylalanine); TYR and Tyr(tyrosine); PBMC (peripheral blood mononuclear cells); LB (Luria broth);MeOH (methanol); Athens Research (Athens Research Technology, Athens,Ga.); ProSpec (ProSpec Tany Technogene, East Brunswick, N.J.);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Ram Scientific (RamScientific, Inc., Yonkers, N.Y.); Pall Corp. (Pall, Corp., Pt.Washington, N.Y.); Millipore (Millipore, Corp., Billerica Mass.); Difco(Difco Laboratories, BD Diagnostic Systems, Detroit, Mich.); MolecularDevices (Molecular Devices, LLC, Sunnyvale, Calif.); Kuhner (AdolfKuhner, AG, Basel, Switzerland); Microfluidics (Microfluidics Corp.,Westwood, Mass.); Thermotron (Thermotron, Inc., Holland, Mich.);Cambridge isotope Laboratories. (Cambridge Isotope Laboratories, Inc.Tewksbury, Mass.); Applied Biosystems (Applied Biosystems, part of LifeTechnologies, Corp., Grand Island, N.Y.); Greiner Bio-One (GreinerBio-One North America, Monroe, N.C.); Agilent (Agilent Technologies,Inc., Santa Clara, Calif.); Thermo Scientific (part of Thermo FisherScientific, Waltham, Mass.); Corning (Corning, Inc., Palo Alto, Calif.);Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo LifeSciences, Inc., Farmingdale, N.Y.); GE Healthcare (GE HealthcareBio-Sciences, Piscataway, N.J.); Pierce (Pierce Biotechnology (now partof Thermo Fisher Scientific), Rockford, Ill.); Phenomenex (Phenomenex,Inc., Torrance, Calif.); Optimal (Optimal Biotech Group, Belmont,Calif.); and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.).

EXAMPLE 1 Synthesis and Assaying of TAL Enzymes with Tyrosine AmmoniaLyase (TAL) Activity

In this Example, methods used in the synthesis and assaying of TALenzymes for tyrosine ammonia lyase activity are described.

TAL Gene Acquisition and Construction of Expression Vectors

Polynucleotide sequences encoding phenylalanine and tyrosine ammonialyases from Stanieria cyanosphaera (SEQ ID NO: 2), Chroogloeocystissiderophila (SEQ ID NO: 4), Flavobacterium johnsoniae (SEQ ID NO: 6),and Rhodotorula glutinis (SEQ ID NO: 8) were synthesized as the genes ofSEQ ID NOS: 1, 3, 5, and 7, respectively. These synthetic genes werecloned into a pCK110900 vector system (See e.g., U.S. Pat. No. 7,629,157and US Pat. Appln. Publn. Nos. 2016/0244787, and 2006/0195947, all ofwhich are incorporated by reference), and subsequently expressed in anE. coli strain derived from W3110. In some embodiments, expressionvectors lacking antimicrobial resistance markers find use.

High-Throughput (HTP) Growth of Cultures Expressing TAL Enzymes

Transformed E. coli cells were selected by plating onto LB agar platescontaining 1% glucose and 30 μg/mL chloramphenicol. After overnightincubation at 37° C., colonies were picked onto NUNCTM(Thermo-Scientific) 96-well shallow flat bottom plates filled with 180μL/well LB-medium supplemented with 1% glucose and 30 μg/mLchloramphenicol. Cultures were allowed to grow overnight for 18-20 hoursin a shaker (200 rpm, 30° C., and 85% relative humidity; Kuhner).Overnight growth samples (20 μL) were transferred into Costar 96-welldeep plates filled with 380 μL of TB) supplemented with 30 μg/mLchloramphenicol. Cultures were incubated for 2-3 hours in a shaker (250rpm, 30° C., and 85% relative humidity; Kuhner) and then induced with 40μL of 10 mM IPTG in sterile water and incubated overnight for 20-24hours in a shaker (250 rpm, 30° C., and 85% relative humidity; Kuhner).Cells were pelleted (4000 rpm×10 min), supernatants were discarded, andthe cells were frozen at −80° C. prior to analysis.

Lysis of HTP Cell Pellets

E. coli cell pellets were lysed with 250 μL of lysis buffer (1 mg/mllysozyme+0.5 g/L PMBS in 20 mM sodium phosphate pH 8, with 150 mM NaCl).The mixture was agitated for 2 hours at room temperature, and pelleted(4000 rpm×10 min), after which the clarified lysates were used in HTPassays described herein.

HTP Activity Analysis of Clarified Lysates

TAL variant activity on tyrosine or phenylalanine was assayed bymeasuring the formation of coumaric or cinnamic acid, respectively, asdetermined by the change in absorbance at 290 nm over time. Reactionswere prepared by the addition of 160 μL of 2.5 mM tyrosine orphenylalanine dissolved in 200 mM sodium phosphate and 40 μL ofclarified lysate to a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and the activity was determined bytracking the absorbance at 290 nm over time (every 14-37s over 10 min)using a SPECTRAMAX® Plus 384 or a SPECTRAMAX® 190 (Molecular Devices)absorbance microplate reader. Activity on tyrosine was detected for SEQID NOS: 4, 2, 6, and 8 as shown in Table 1-1. Some enzymes also haddetectable activity on phenylalanine.

TABLE 1-1 Activity of TAL Variants on Tyrosine and Phenylalanine UnderVarious Conditions¹ SEQ ID NO: (nt/aa) 2 mM Tyr Activity 2 mM PheActivity 1/2 ++++ 3/4 ++ 5/6 +++ 7/8 + + ¹Levels of initial activity(milli-Absorbance Units/min) are defined as follows: ““+”” > 10; “++” >20; “+++” > 50; and “++++” > 100.

EXAMPLE 2 Shake Flask Powder (SFP) Production and CharacterizationAssays for TAL and TAL Variants

In this Example, methods used for the preparation and characterizationof shake flask powders of TAL enzymes with tyrosine ammonia lyaseactivity are described.

Production of Shake Flask Powders (SFP)

E. coli cultures transformed with plasmids containing TAL variants wereplated onto LBagar plates with 1% glucose and in some instances, 30μg/mL chloramphenicol, and grown overnight at 37° C. A single colonyfrom each culture was transferred to 5 mL of Luria Broth (LB) with 1%glucose and 30 μg/mL chloramphenicol, where appropriate. The cultureswere grown for 18 h at 30° C., 250 rpm, and subcultured approximately1:50 into 250 ml of TB with 30 μg/mL of chloramphenicol. The cultureswere grown for approximately 3-4 h at 30° C., 250 rpm, to an OD₆₀₀ of0.6-0.8, and induced with 1 mM of IPTG. The cultures were grown for 20 hat 30° C., 250 rpm. Cells were harvested by centrifugation (7000 rpm×10min, 4° C.), and the supernatants were discarded. The pellets wereresuspended in 30 mL of 20 mM sodium phosphate, pH 8.0, with 150 mM NaClin some instances, and lysed using a single pass through amicrofluidizer (Microfluidics), at 110 psi. The lysates were pelleted(10,000×rpm, 30 min, 4° C.), and the supernatants were heated for 1-2hours at 60-65° C. in a water bath before being pelleted again(10,000×rpm, 1 h, 4° C.). The resulting supernatants were then frozenand lyophilized to generate powders containing the expressed enzymes.

SFP Characterization Assay for Activity on Tyrosine and Phenylalanine

Shake flask powders were reconstituted to provide 10-20 g/L powder andwere serially diluted to 0.08-10 g/L. Then, 20 μL of these stocks wereadded to 180 μL of 2.5 mM tyrosine or phenylalanine dissolved in 200 mMsodium phosphate, pH 7.0, in a 96-well UV-STAR® plate (Greiner Bio-One).The reactions were mixed briefly, and the activity was determined bytracking the absorbance at 290 nm over time (every 14-37 s over 5 min)using a SPECTRAMAX® Plus 384 or a SPECTRAMAX® 190 (Molecular Devices)absorbance microplate reader. Results for SEQ ID NOS: 2 and 6 are shownin Table 2-1.

In some cases, TAL enzyme activity on phenylalanine was measured overlonger periods of time. Reactions were prepared by adding 20 μL ofdiluted shake flask powder solution to 180 μL of 2.5 or 12.5 mMphenylalanine dissolved in 200 mM sodium phosphate, pH 7.0. Thereactions were mixed briefly, and an initial absorbance was determinedby diluting the assay 10-fold in water and measuring the absorbance at290 nm using a SPECTRAMAX® Plus 384 or a SPECTRAMAX® 190 (MolecularDevices) absorbance microplate reader. After incubation at 37° C. for1-4 h in a THERMOTRON® (INFORS) titre-plate shaker (400 rpm), the finalabsorbance was measured in a similar manner Activity was determined bysubtracting the initial from the final absorbance.

SFP Characterization Assay for Resistance to Proteases

To evaluate the relative stability of evolved enzymes to representativeproteases, a mix of porcine trypsin (Sigma Aldrich) and bovinechymotrypsin (Sigma Aldrich) were dissolved in 100 mM sodium phosphate,pH 7.0, to a concentration of 8 g/L each and serially diluted 2-fold.Then, 0.5-1 g/L TAL SFP were incubated with 0-4 g/L at 37° C. for 1-2 hat 400 rpm in a THERMOTRON® (INFORS) titre-plate shaker. Afterincubation, 20-40 μL of the protease-treated SFP was added to 160-180 μLof 2.5 mM tyrosine dissolved in 200 mM sodium phosphate, pH 7.0, andenzyme initial activity was measured as described above. Proteaseresistance was calculated as a percentage of residual activity againstthe control (i.e., no protease treatment) sample. Results for SEQ IDNOS: 2 and 6 are shown in Table 2-1.

SFP Characterization Assay for pH Tolerance

To evaluate the relative tolerance of evolved enzymes to acidic pH, SFPswere reconstituted to provide 10 g/L powder and were diluted 10-foldinto McIlvaine buffer, pH 4.0-7.0. The mixture was incubated for 1 h at37° C. for 1-1.5 h at 400 rpm in a THERMOTRON® (INFORS) titre-plateshaker. After incubation, the enzyme solutions were briefly centrifuged,and 20 μL of the solutions were added to 180 μL of 2.5 mM tyrosinedissolved in 200 mM sodium phosphate, pH 7.0. Enzymatic activity wasdetermined as described above, and pH tolerance was calculated as apercentage of residual activity against the control (i.e., pH 7.0)sample.

SFP Characterization Assay for Determination of Kinetic Parameters

To evaluate if the mutations in the TAL variants had altered kinetics,the Michaelis constant (K_(m)) and maximum velocity (V_(max)) weredetermined for each variant. To assay, 20 μL of diluted TAL variants at1 g/L and 180 μL of 2-fold serially diluted tyrosine (0-2.48 mM tyrosinein 200 mM sodium phosphate, pH 7.0), were added to the wells of a96-well UV-STAR® plate (Greiner Bio-One). The reactions were mixedbriefly, and the activity was determined by tracking the absorbance at290 nm over time (every 14-37 s over 5 min) using a SPECTRAMAX® Plus 384or a SPECTRAMAX® 190 (Molecular Devices) absorbance microplate reader.The V_(max) and K_(m) for each tested TAL variant was determined byfitting the data to a Michaelis-Menten equation using non-linearregression. Results for SEQ ID NOS: 2 and 6 are shown in Table 2-1.

SFP Characterization Assay for Thermostability

Improved thermostability is a valuable trait useful in manufacture andstorage of enzyme therapeutics and often occurs as a byproduct of otherstabilization efforts. To assess the relative stability of the variantsproduced during the development of the present invention, thethermostability of the variants was assessed as follows: 100 μL of TALvariants at 1 g/L were incubated for 2 h at 60-80° C. in a thermocycler.After incubation, samples were briefly centrifuged, and 20 μL of theheat-treated SFP was added to 180 μL of 2.5 mM tyrosine dissolved in 200mM sodium phosphate, pH 7.0. Enzymatic activity was determined asdescribed above, and thermostability was calculated as a percentage ofresidual activity against the control (i.e., 60° C.) sample. Results forSEQ ID NOS: 2 and 6 are shown in Table 2-1.

TABLE 2-1 Activity of TAL Variants on Tyrosine and Phenylalanine UnderVarious Conditions SEQ Protease Resistance Thermostability ID NO: TyrVmax Tyr Km (% Residual Activity (% Residual (nt/aa) (mAU/min)¹ (mM)2 at0.5 g/L Protease) Activity at 60° C.) 1/2 +++ + + +++ 5/6 + ++ ++++ ¹TyrVmax is defined as follows: “+” = 0.5-1; “++” > 1; and “+++” > 5. 2TyrKm is defined as follows: “+” = 0.15-0.05; and “++” < 0.05. The %residual activity for protease and thermostability challenges is definedas follows: “+” = 5-50%; “++” > 50%; “+++” > 75%; and “++++” > 90%.

EXAMPLE 3 TAL Variants of SEQ ID NO: 2

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 2, for improved tyrosine activity andphenylalanine activity are described. Directed evolution of the TALencoded by SEQ ID NO: 1, was carried out by constructing libraries ofvariant genes, using methods known in the art. These libraries were thenplated, grown, and screened using the methods described below.

HTP Activity Analysis of Clarified Lysates:

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 1. TAL variant activity on tyrosineand phenylalanine were assayed as described in Example 1, with thefollowing conditions: 20 μL of HTP lysate diluted 10-fold in water wasassayed with 2.2 mM tyrosine for 5 min, 20 μL of undiluted HTP lysatewas assayed with 0.1 mM tyrosine for 10 min, and 50 μL of undiluted HTPlysate was assayed with 10.5 mM phenylalanine for 10 min. The results ofthe assays are shown in Table 3-1. Based on these results, SEQ ID NO: 14was selected for further directed evolution.

TABLE 3-1 Relative Activity of TAL Variants on Tyrosine andPhenylalanine Under Various Conditions (Relative to SEQ ID NO: 2)¹ 2 mMTyr 2 mM Phe SEQ ID FIOPC FIOPC NO: Amino Acid Differences (Relative to(Relative to (nt/aa) (Relative to SEQ ID NO: 2) SEQ ID NO: 2) SEQ ID NO:2)  9/10 N99H/V107A/Y184S + +++ 11/12 S79T/V107S/P410K + ++++ 13/14K95A/V107A/Y184S ++ ++++ 15/16 S79T/P410K + +++ 17/18V107S/A401P/A534S + +++ 19/20 S79T/K95A/V107S + ++++ 21/22 S79T/V107S ++++ 23/24 K95A/Y184S/A534S + +++ 25/26 V107A/Y184S/A534S ++ +++ 27/28K95A/V107S/Y184S/A534S ++ +++ ¹All activities were determined relativeto the reference polypeptide SEQ ID NO: 2. Levels of increased activityare defined as follows: ““+”” = 0.9 to 1.2; “++” > 1.2; “+++” > 2; and“++++” > 4.

EXAMPLE 4 TAL Variants of SEQ ID NO: 14

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 14, for improved tyrosine activity,phenylalanine activity, and protease resistance are described. Directedevolution of the TAL encoded by SEQ ID NO: 13, was carried out byconstructing libraries of variant genes. These libraries were thenplated, grown, and screened using the methods described below.

High-Throughput (HTP) Growth of Cultures Expressing TAL Enzymes

Transformed E. coli cells were selected by plating onto LB agar platescontaining 1% glucose. After overnight incubation at 37° C., colonieswere picked onto NUNC™ (Thermo-Scientific) 96-well shallow flat bottomplates filled with 180 μL/well LB-medium supplemented with 1% glucose.Cultures were allowed to grow overnight for 18-20 hours in a shaker (200rpm, 30° C., and 85% relative humidity; Kuhner). Overnight growthsamples (20 μL) were transferred into Costar 96-well deep plates filledwith 380 μL of TB supplemented with 30 μg/mL chloramphenicol. Cultureswere incubated for 2-3 hours in a shaker (250 rpm, 30° C., and 85%relative humidity; Kuhner) and then induced with 40 μL of 10 mM IPTG insterile water and incubated overnight for 20-24 hours in a shaker (250rpm, 30° C., and 85% relative humidity; Kuhner). Cells were pelleted(4000 rpm×10 min), supernatants were discarded, and cells were frozen at−80° C. prior to analysis.

Lysis of HTP Cell Pellets

E. coli cell pellets were lysed with 400 μL of lysis buffer (1 mg/mllysozyme+0.5 g/L PMBS in 20 mM sodium phosphate pH 8, with 150 mM NaCl).The mixture was agitated for 1.5-2 hours at room temperature, andpelleted (4000 rpm×10 min) after which the clarified lysates were usedin HTP assays or preincubated for 2 h at 65° C. in a THERMOTRON®(INFORS) titre-plate shaker (400 rpm). The heat-treated lysates werepelleted (4000 rpm×10 min), and the supernatants were used in HTPassays.

HTP Activity Analysis of Clarified Lysates

TAL variant activity was assayed by measuring the formation of coumaricacid as determined by the change in absorbance at 290 nm over time.Reactions were prepared by the addition of 180 μL of 200 mM sodiumphosphate, 0.125 or 2.5 mM tyrosine, pH 7.0, and 20 μL of heat-treatedclarified lysate (undiluted for 0.125 mM tyrosine or diluted 5-fold for2.5 mM tyrosine) to a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and the activity was determined bytracking the absorbance at 290 nm over time (every 14-37 s over 5 min)using a SPECTRA® Plus 384 or a SPECTRAMAX® 190 (Molecular Devices)absorbance microplate reader. The results of these assays are shown inTable 4-1, and SEQ ID NO: 86 was selected for further directedevolution.

TAL variant activity on phenylalanine was assayed by measuring theformation of cinnamic acid as determined by the change in absorbance at290 nm over time. Reactions were prepared by adding 160 μL of 200 mMsodium phosphate, 12 mM phenylalanine, pH 7.0, and 40 μL of heat-treatedclarified lysate to a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and an initial absorbance was determinedby measuring the absorbance at 290 nm using a SPECTRAMAX® Plus 384 or aSPECTRAMAX® 190 (Molecular Devices) absorbance microplate reader. Afterincubation at 37° C. for 1.5-3 h in a THERMOTRON® (INFORS) titre-plateshaker (400 rpm), the final absorbance was measured in a similarfashion. Activity was determined by subtracting the initial from thefinal absorbance, and results are shown in Table 4-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin to simulatethe environment of the lower intestine. First, 50 μL of protease mix(0.25 mg/mL chymotrypsin (Sigma Aldrich), 0.25 mg/mL trypsin (SigmaAldrich)) in 100 mM sodium phosphate pH 7.0 and 50 μL of heat-treatedclarified lysate were added to a 96-well round bottom plate (Costar).The plates were sealed and incubated for 1 hour at 37° C. in aTHERMOTRON® (INFORS) titre-plate shaker (400 rpm) prior to analysis.Residual activity was determined by adding 40 μL of the protease-treatedlysate to 160 μL of 200 mM sodium phosphate, 2.5 mM tyrosine, pH 7.0, ina 96-well UV-STAR® plate (Greiner Bio-One). The reactions were mixedbriefly, and activity was determined as described in Example 3. Theresults of this assay are shown in Table 4-1.

TABLE 4-1 Relative Activity of TAL Variants on Tyrosine andPhenylalanine Under Various Conditions (Relative to SEQ ID NO: 14)¹ SEQID NO: Amino Acid Differences 2 mM Tyr Protease 0.1 mM Tyr 10 mM Phe(nt/aa) (Relative to SEQ ID NO: 14) FIOPC FIOPC FIOPC FIOPC 29/30K33D/K34E/Q37S/T90S/K554Q ++ +++ ++ ++ 31/32 A289R/K554Q ++ ++ ++ ++33/34 K33D/Q66N/A289R/K554Q ++ +++ ++ ++ 35/36 K34E/L46R +++ +++ ++ ++37/38 K33D/Q66N/A289R ++ ++ ++ ++ 39/40 K33D/K34E/T90S/A289R/ ++ +++ ++++ K554Q 41/42 K33D/Q66N ++ ++ ++ ++ 43/44 K34E/T90S/A289R/K554Q ++ ++++ ++ 45/46 K34E/K56G/T90S/A289R ++ ++ ++ ++ 47/48Q66N/T90S/Q279D/A289R/ ++ ++ ++ ++ K554Q 49/50 T90S/A289R/K554Q ++ ++ ++++ 51/52 K34E/Q66N/A289R ++ ++ ++ ++ 53/54 K33D/K34E/Q37S/L46R/Q279S/ +++++ ++ ++ K554Q 55/56 K33D/K34E/L46R/Q66N/T90S/ ++ ++ ++ ++ A289R 57/58K34E/Q37R/L46R/A289R ++ +++ ++ ++ 59/60 K33D/K34E/Q279S/A289R ++ +++ ++++ 61/62 K34E/K56G/T90S ++ ++ ++ ++ 63/64 K34E/Q37S/Q66N/Q279S/ ++ ++ ++++ A289R 65/66 Q279S/A289R/K554Q ++ ++ ++ ++ 67/68 T90S/A289R/A519P ++++ ++ + 69/70 K34E/T90S/A519P ++ ++ ++ ++ 71/72 Q66N/T90S/A289R/K554Q ++++ ++ ++ 73/74 A289R/A519P/K554Q ++ ++ ++ ++ 75/76L46R/Q66N/A289R/A519P/ ++ ++ ++ ++ K554Q 77/78 S79T/E304G/L361M ++ +++++ + 79/80 S79T/Q111K/N531D ++ +++ ++ ++ 81/82 S79T/S407V/L461M ++ +++ +++ 83/84 S79T/Q111K/Y234H/L361M/ ++ ++++ + ++ C500T 85/86S70K/S79T/Y234H/L361M/ ++ ++++ + ++ S407V 87/88 E304G/L361M ++ +++ ++ +89/90 S79T/E304G/L461M/N53ID ++ +++ ++ + 91/92 S70K/E304G/S407V/L461M +++++ ++ ++ 93/94 S79T/Q111K/Y234H/N531D ++ +++ ++ ++ 95/96A36V/S407V/L461M/C500T/ ++ ++ ++ ++ N531D 97/98 A36V/Q111K/E304G/L461M/++ ++ ++ + N531D  99/100 L461M/C500T ++ ++ ++ ++ 101/102S70K/Q111K/L461M/C500T/ ++ +++ ++ ++ N531D 103/104 L361M/C500Q/N531D ++++ ++ + 105/106 A36V/S79T/S407V/L461M/ ++ ++++ ++ ++ C500T/N531D 107/108A36V/S70K/Y234H/R302M/ ++ +++ ++ L461M/C500T 109/110 A36V/S79T + +++ +++ 111/112 A36V/S79T/E304G/S407V + +++ ++ + 113/114 N531D ++ ++ ++ ++115/116 S79T/Q111K/L361M ++ ++++ ++ ++ 117/118 S548R ++ +++ ++ ++119/120 A544E ++ +++ ++ ++ 121/122 K28G +++ +++ +++ ++ 123/124 E522K ++++++ ++ ++ 125/126 A540R ++ +++ ++ ++ 127/128 A544G ++ +++ ++ ++ 129/130T547S ++ +++ ++ ++ 131/132 Q558A ++ ++ ++ ++ 133/134 A544S ++ +++ +++ ++135/136 R524Y ++ ++ ++ ++ 137/138 L295Q +++ ++ +++ + 139/140 T547C ++ ++++ ++ 141/142 L541A ++ +++ ++ ++ 143/144 I491V ++ ++ ++ ++ 145/146 E522G++ +++ ++ ++ 147/148 Q66S ++ ++ ++ ++ 149/150 S548Q ++ ++ ++ ++ 151/152K518E ++ +++ ++ ++ 153/154 P29E ++ +++ ++ ++ 155/156 E522R ++ ++++ ++ ++157/158 T547Q +++ +++ ++ ++ 159/160 S548T ++ +++ ++ ++ 161/162 K490I +++++ +++ ++ 163/164 K518V +++ +++ ++ ++ 165/166 V88Q +++ ++ +++ ++ 167/168E522V ++ +++ ++ ++ 169/170 K201R ++ ++ ++ ++ 171/172 K554S ++ +++ ++ ++173/174 K28N ++ +++ ++ ++ 175/176 K28C ++ +++ ++ ++ 177/178 A278S ++ ++++ ++ 179/180 I491L ++ ++ ++ ++ 181/182 K33N ++ +++ ++ ++ 183/184 P29R++ ++ ++ ++ 185/186 A540E ++ +++ ++ ++ 187/188 R524Q +++ +++ +++ ++189/190 T547A +++ +++ ++ ++ 191/192 H131M +++ ++ +++ ++ 193/194 S548K +++++ ++ ++ 195/196 E522Y ++ ++ ++ ++ 197/198 P29A ++ ++ ++ ++ 199/200K554Y ++ ++ ++ + 201/202 V560R ++ ++ ++ ++ 203/204 K490A +++ ++ +++ ++205/206 T49N ++ +++ ++ ++ 207/208 K28A ++ ++ ++ ++ 209/210 T547K ++ +++++ ++ 211/212 A519Q ++ +++ +++ ++ 213/214 L46R ++ ++ ++ ++ 215/216 R524A++ ++ ++ ++ 217/218 Q558H ++ ++ ++ ++ 219/220 R524T ++ ++ ++ ++ 221/222Q66R +++ +++ +++ ++ 223/224 V88E +++ ++ ++ + 225/226 Q37A ++ ++ ++ ++227/228 S548A ++ ++ ++ ++ 229/230 V560T ++ ++ ++ ++ 231/232 P272S ++ ++++ ++ 233/234 A519L ++ ++ ++ ++ 235/236 K490V ++ ++ ++ ++ 237/238 K554R++ +++ ++ ++ 239/240 L510K ++ +++ ++ ++ 241/242 T547G ++ ++ ++ ++243/244 S548G ++ +++ ++ ++ 245/246 R524L +++ +++ +++ ++ 247/248 Q558S+++ +++ ++ ++ 249/250 T547R +++ +++ +++ ++ 251/252 N557E +++ +++ +++ +++253/254 T49D +++ +++ +++ +++ 255/256 E72T +++ ++ ++ + 257/258 Q558D ++++++ +++ +++ 259/260 V560G ++ ++ ++ ++ 261/262 E523D ++ +++ ++ ++ 263/264P29G ++ ++ ++ +++ 265/266 E523I + + + + 267/268 A519E +++ +++ ++ ++269/270 E523S ++ ++ ++ ++ 271/272 K554E ++ +++ ++ ++ 273/274 L46T ++ ++++ ++ 275/276 R524I +++ ++ ++ ++ 277/278 Q305H +++ ++ +++ ++ 279/280V560M ++ +++ ++ ++ 281/282 Q142R ++ ++ ++ ++ 283/284 A519D ++ ++ ++ ++285/286 E523A ++ ++ ++ ++ 287/288 E523H ++ ++ ++ ++ 289/290 H131S ++ ++++ ++ 291/292 K28T ++ ++ ++ ++ 293/294 H131W ++ ++ ++ ++ 295/296 A69S ++++ ++ ++ 297/298 K490S ++ + ++ ++ 299/300 P29W ++ ++ ++ ++ 301/302 A519P++ ++ ++ ++ 303/304 Q37D +++ +++ +++ +++ 305/306 H131G + +++ ++ ++++307/308 A350G +++ +++ ++ ++ 309/310 K56R ++ ++ ++ ++ 311/312 E198R + +++ + 313/314 S521D +++ +++ ++ ++ 315/316 Q499T ++ ++ ++ ++ 317/318 K56G+++ +++ +++ +++ 319/320 N557R ++ ++ ++ ++ 321/322 A544R +++ +++ ++ ++¹All activities were determined relative to the reference polypeptideSEQ ID NO: 14. Levels of increased activity are defined as follows:““+”” = 0.9 to 1.1; “++” > 1.1; “+++” > 2; and “++++” > 3.

EXAMPLE 5 TAL Variants of SEQ ID NO: 86

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 86 for improved tyrosine activity, phenylalanineactivity, protease resistance, and low pH tolerance are described.Directed evolution of the TAL encoded by SEQ ID NO: 85, was carried outby constructing libraries of variant genes using methods known in theart. These libraries were then plated, grown, and screened using themethods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosineand phenylalanine were assayed as described in Example 4, with thefollowing conditions: 20 μL of HTP lysate diluted 4- or 5-fold in waterwas assayed with 2.2 mM tyrosine for 5 min, 20 μL of undiluted HTPlysate was assayed with 0.1 mM tyrosine for 5 min, and 20 μL ofundiluted HTP lysate was assayed with 10.8 mM phenylalanine for 2 h. Theresults of the assays are shown in Table 5-1. SEQ ID NO: 334 wasselected for further directed evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin, and 50 μL ofheat-treated clarified lysate were incubated with 50 μL of protease mixconsisting of 8 mg/mL chymotrypsin (Sigma Aldrich) and 8 mg/mL trypsin(Sigma Aldrich) in 100 mM sodium phosphate pH 7.0. Residual activity ontyrosine was measured as described in Example 4, and the results of thisassay are shown in Table 5-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer

TAL variants were challenged with acidic buffer to simulate the gastricenvironment. First, 50 μL of McIlvaine buffer pH 4 or 4.2, and 50 μL ofheat-treated clarified lysate were added to a 96-well round bottom plate(Costar). The plates were sealed and incubated for 1 hour at 37° C. in aTHERMOTRON® (INFORS) titre-plate shaker (400 rpm) prior to analysis.After incubation, the lysate solutions were briefly centrifuged, andresidual activity was determined by adding 40 μL of the acidicbuffer-treated lysate to 160 μL of 200 mM sodium phosphate, 2.5 mMtyrosine, pH 7.0, in a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and activity was determined as describedin Example 4. The results of this assay are shown in Table 5-1.

TABLE 5-1 Relative Activity of TAL Variants on Tyrosine andPhenylalanine Under Various Conditions (Relative to SEQ ID NO: 86)¹ SEQID Amino Acid Differences 2 mM 0.1 mM 10 mM NO: (Relative to TyrProtease Tyr pH 4 Phe (nt/aa) SEQ ID NO: 86) FIOPC FIOPC FIOPC FIOPCFIOPC 323/324 K33D/K34E/Q66N/A289R/ ++ ++ ++ ++++ ++ E522K/N531D 325/326K33D/K34E/K70S/H234Y/ ++ ++ ++ ++++ ++ A289R/A519P 327/328K34E/Q66N/K70S/H234Y/ +++ +++ ++ ++++ ++ K518V/A519P/T547Q 329/330K33N/K34E/T49N/H234Y/ ++ ++ ++ ++++ ++ N531D/T547Q/K554Q 331/332K34E/Q66N/N531D/K554Q +++ +++ ++ +++ ++ 333/334 K34E/A289R/E522K/A544E/+++ ++ +++ ++++ ++ K554Q 335/336 K33D/K34E/K70S/H234Y/ ++ ++ ++ +++ ++K518V/A519P/T547Q/K554Q 337/338 Q66N/K70S/H234Y ++ ++ + +++ + 339/340K34E/H234Y/E522K/K554Q ++ ++ +++ ++++ ++ 341/342 K34E/K70S/A289R/E522K++ ++ ++ ++ + 343/344 K34E/A289R ++ ++ ++ +++ ++ 345/346H234Y/N531D/A544E/T547Q ++ ++ ++ +++ ++ 347/348K33D/K34E/Q66N/K70S/A519P/ +++ +++ + +++ ++ N531D 349/350K34E/T49N/Q66N/N531D +++ ++ ++ +++ ++ 351/352 K34E/K70S/H234Y/A289R/ ++++ ++ ++++ + E522K 353/354 K28N/K34E/T49N/K70S/H234Y/ +++ ++ ++ +++ ++A289R/A544E/K554Q 355/356 K33D/K34E/K70S/A289R/ +++ ++ ++ +++ ++E522K/K554Q 357/358 K33D/K34E/A289R/E522K/ +++ ++ ++ ++++ ++ N531D359/360 K34E/K70S/H234Y/K554Q +++ ++ ++ ++++ ++ 361/362K34E/T49N/A289R/A544E/ +++ ++ +++ +++ ++ T547Q 363/364K34E/A289R/E522K/N531D +++ +++ ++ ++++ ++ 365/366 K34E/Q66N/K554Q +++ +++++ +++ ++ 367/368 K34E/A544E ++ ++ +++ +++ ++ 369/370K34E/T49N/H234Y/N531D/ +++ +++ +++ ++++ ++ K554Q 371/372 K34E/E522K ++++ +++ +++ ++ ¹All activities were determined relative to the referencepolypeptide SEQ ID NO: 86. Levels of increased activity are defined asfollows: ““+”” = 0.9 to 1.2; “++” > 1.2; “+++” > 2; and “++++” > 3.

EXAMPLE 6 TAL Variants of SEQ ID NO: 334

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 334 for improved tyrosine activity,phenylalanine activity, protease resistance, and low pH tolerance aredescribed. Directed evolution of the TAL encoded by SEQ ID NO: 333, wascarried out by constructing libraries of variant genes. These librarieswere then plated, grown, and screened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 4-10-fold in water was assayed with 2.2 mMtyrosine for 5 min and 20 μL of undiluted HTP lysate was assayed with0.1 mM tyrosine for 5 min. TAL variant activity on phenylalanine wasassayed as described in Examples 1 and 4, with the following conditions:20 μL of undiluted HTP lysate was assayed with 2.2 mM phenylalanine for5 min at room temperature or for 1-3 h at 37° C. The results of theassays are shown in Table 6-1. SEQ ID NO: 388 was selected for furtherdirected evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 4, and 50 μL of heat-treated clarified lysate was incubatedwith either 50 μL 8 g/L protease mix for 1 h at 37° C. or with 4 g/Lprotease mix for 2 h at 37° C. Residual activity was determined byadding 20 μL of the protease-treated lysate to 180 μL of 200 mM sodiumphosphate, 2.5 mM tyrosine, pH 7.0, in a 96-well UV-Star® plate (GreinerBio-One). The reactions were mixed briefly, and activity was determinedas described in Example 4. The results of this assay are shown in Table6-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer

TAL variants were challenged with acidic buffer as described in Example5, and 50 μL of heat-treated clarified lysate was incubated with 50 μLMcIlvaine buffer pH 4. Residual activity was determined as described inExample 5, and the results of this assay are shown in Table 6-1.

TABLE 6-1 Relative Activity of TAL Variants on Tyrosine andPhenylalanine Under Various Conditions (Relative to SEQ ID NO: 334)¹ SEQID Amino Acid Differences 2 mM 0.1 mM 2 mM NO: (Relative to Tyr ProteaseTyr pH 4 Phe (nt/aa) SEQ ID NO: 334) FIOPC FIOPC FIOPC FIOPC FIOPC373/374 I336V ++ ++ + ++ + 375/376 L506R ++ + + + + 377/378A43T/S59A/L93Q/L506R + + + + + 379/380 Q237R/N238V/L506R + + + + +381/382 A65N/I336V ++ + + + + 383/384 P306L/L506R ++ + + ++ + 385/386A43T/P306L ++ + ++ + 387/388 L93Q/L506R + ++ + + + 389/390 P306L ++ + +++ + 391/392 A43T/P306L/L506R + + + + 393/394 R103W/P306L/L506R + + +++ + 395/396 K56G/H234Y/C500T + ++ + ++ + 397/398 Q37D/T49N/K56G/C500T/++ ++ + + + R524Q 399/400 Q66N ++ ++ ++ ++ + 401/402Q37D/T49N/Q111K/R524Q ++ + ++ ++ + 403/404 Q66N/Q111K/H234Y/C500T +++ + + + 405/406 T49N/K56G/Q66N/Q111K/ + + + ++ +T153N/H234Y/C500T/R524Q 407/408 Q37D/Q111K ++ ++ + + + 409/410 H234Y ++++ ++ + 411/412 Q37D/H234Y + + + ++ + 413/414 Q37D/T49N/Q66N/Q111K/ +++ + + + H234Y 415/416 Q111K/M361L ++ + ++ + 417/418 Q558D + ++ + ++ +419/420 T49N/Q66N ++ + + ++ + 421/422 Q37D/T49N/K56G/Q66N/ + + + ++ +Q111K 423/424 M361L/R524Q/N557E/Q558D + + + + + 425/426Q37D/T49N/K56G/H234Y/ ++ + + ++ + N557E 427/428 Q37D/Q111K/M361L/C500T/++ ++ ++ ++ + N557E/Q558D 429/430 Q111K/H234Y/S548K/Q558D ++ ++ ++ + +431/432 H234Y/R524Q ++ ++ ++ + + 433/434 K56G/Q111K/H234Y ++ + ++ ++ +435/436 T421S ++ ++ + + 437/438 A107T/G417A/T421A + + 439/440G417A/T421S ++ ++ + + ++ 441/442 A107T ++ + ++ ++ + 443/444R103W/T421S + + + ++ 445/446 A172S + 447/448R103W/A107T/G417A/T421S + + + ++ + 449/450 R103W + + + + 451/452Y104F/H105M ++++ 453/454 A504R + + + + + 455/456 S328A + + + + + 457/458E145V + + + + + 459/460 Q471Y + + + + 461/462 R103L + + + + ++ 463/464G549D + + + + + 465/466 C61V ++ + + 467/468 E304N ++ + 469/470V407T + + + + 471/472 L211K + + 473/474 H155S + + + + + 475/476T146K + + ++ + + 477/478 A95K + + + + 479/480 A65M + + 481/482A286Q + + + + + 483/484 A95V + ++ + + 485/486 T31S + + + + 487/488P197T + + + 489/490 V407K + + + + 491/492 H155L + + + + 493/494A286R + + + 495/496 L506V + + ++ + + ¹All activities were determinedrelative to reference polypeptide SEQ ID NO: 334. Levels of increasedactivity are defined as follows: “+” = 0.9 to 1.2; “++” > 1.2; “+++” >2; and “++++” > 3.

EXAMPLE 7 TAL Variants of SEQ ID NO: 388

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 388 for improved tyrosine activity,phenylalanine activity, protease resistance, and low pH tolerance aredescribed. Directed evolution of the TAL encoded by SEQ ID NO: 387, wascarried out by constructing libraries of variant genes. These librarieswere then plated, grown, and screened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min and 20 μL of undiluted HTP lysate was assayed with0.1 mM tyrosine for 5 min. TAL variant activity on phenylalanine wasassayed as described in Example 4 with the following conditions: 20 μLof undiluted HTP lysate was assayed with 2.2 mM phenylalanine for 5 minat room temperature or for 2 h at 37° C. The results of the assays areshown in Table 7-1. SEQ ID NO: 604 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 4, and 50 μL of heat-treated clarified lysate was incubatedwith 50 μL 8 g/L protease mix for 2 h at 37° C. Residual activity wasdetermined as described in Example 6. The results of this assay areshown in Table 7-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer

TAL variants were challenged with acidic buffer as described in Example5, and 50 μL of heat-treated clarified lysate was incubated with 50 μLMcIlvaine buffer pH 4. Residual activity was determined by adding 20 μLof the acidic buffer-treated lysate to 180 μL of 200 mM sodiumphosphate, 2.5 mM tyrosine, pH 7.0, in a 96-well UV-Star® plate (GreinerBio-One). The reactions were mixed briefly, and activity was determinedas described in Example 4. The results of this assay are shown in Table7-1.

TABLE 7-1 Relative Activity of TAL Variants on Tyrosine andPhenylalanine Under Various Conditions (Relative to SEQ ID NO: 388)¹ SEQID Amino Acid Differences 2 mM 0.1 mM 2 mM NO: (Relative to Tyr ProteaseTyr pH 4 Phe (nt/aa) ID NO: 388) FIOPC FIOPC FIOPC FIOPC FIOPC 497/498Q66N/R103L/E145V/H234Y/ ++ ++ ++ ++++ ++ T421S 499/500 S59A/E145V ++ ++++ ++++ ++ 501/502 T49N/S59A/Q66N/Q111K/ ++ +++ ++ ++++ + E145V/H234Y503/504 Q111K/E145V/H234Y ++ ++ ++ ++++ + 505/506 K56G/Q66N/E145V/C500T++ ++ ++ ++++ + 507/508 K56G/S59A/Q66N/H234Y ++ +++ ++ ++++ ++ 509/510R103L/Q111K/E145V/H234Y/ ++ ++ ++ ++++ ++ T421S 511/512 Q111K/H234Y ++++ ++ ++++ ++ 513/514 K56G/S59A/Q111K/E145V/ ++ ++ ++ ++++ ++H234Y/T421S 515/516 T49N/K56G/S59A/H234Y/ ++ +++ ++ ++++ ++ C500T517/518 S59A/H234Y ++ ++ ++ ++++ ++ 519/520 E145V/H234Y/C500T ++ ++ ++++++ + 521/522 K56G/R103L/Q111K/E145V/ ++ +++ ++ ++++ ++ H234Y/C500T523/524 Q37D/R103L/Q111K/E145V/ ++ ++ ++ ++++ ++ T421S/C500T/R524Q525/526 K56G/S59A/Q111K/E145V ++ ++ ++ ++++ + 527/528S59A/R103L/E145V/H234Y/ ++ ++ ++ ++++ ++ T421S/C500T 529/530T49N/S59A/E145V/H234Y/ ++ ++ ++ ++++ ++ T421S 531/532Q37D/T49N/K56G/S59A/ ++ +++ ++ ++ ++ E145V 533/534S59A/R103L/H234Y/T421S ++ ++ ++ ++++ ++ 535/536 C61V/V407T + ++ + ++537/538 A286Q/S328A/V407T/A504R/ ++ ++ ++ ++++ + G549D 539/540C61V/S328A/A504R + ++ + ++++ ++ 541/542 A95K/V407T ++ ++ ++ + 543/544T31S/A95K/A286Q/A504R/ ++ ++ + ++++ + G549D 545/546C61V/A95K/S328A/V407T ++ +++ ++ ++ + 547/548 T31S/A95K/S328A/A504R ++ ++++ + + 549/550 C61V/A95K/V407T ++ +++ ++ ++ ++ 551/552 G549D ++ ++ +++++ ++ 553/554 A95K/A286Q ++ ++ ++ + 555/556 C61V/A504R + ++ + ++++ ++557/558 A95K/A286Q/S328A/A504R ++ ++ ++ ++++ + 559/560A95K/H155S/A286Q/G549D ++ ++ ++ ++++ + 561/562 T31S/A95K/H155S/A286Q/ ++++ ++ ++++ + A504R/G549D 563/564 T31S/A95K/G549D ++ ++ ++ ++++ + 565/566C61V/A95K/A504R/T547Q ++ +++ ++ +++ ++ 567/568 A95K/H155S/A286Q/S328A/++ ++ ++ +++ + V407T/A504R 569/570 T31S/A95K/A504R ++ ++ ++ ++ + 571/572A95K/A286Q/T547Q ++ ++ ++ + 573/574 C61V/S328A/A504R/G549D + +++ + ++++++ 575/576 T31S/C61V/A95K/Q471Y/ ++ +++ ++ ++++ ++ A504R 577/578C61V/A286Q/V407T + +++ ++ ++ ++ 579/580 A95K/S328A/A504R ++ ++ ++ ++ +581/582 A95K/V407T/A504R ++ ++ ++ ++ + 583/584 T31S/C61V/V407T/A504R +++ + ++ ++ 585/586 A95K/G549D ++ +++ ++ ++++ + 587/588T31S/A95K/A286Q/A504R ++ ++ ++ ++ + 589/590 T31S/A95K/Q237K/A504R/ +++++ ++ ++++ + T547Q/G549D 591/592 C61V + +++ + ++ ++ 593/594C61V/A95K/S328A/G549D ++ +++ ++ ++++ ++ 595/596 C61V/A95K/A286Q ++ +++++ + ++ 597/598 C61V/A95K/A286Q/S328A/ ++ +++ ++ ++++ ++ A504R 599/600C61V/A95K/H155S/A286Q/ ++ ++ ++ ++++ + V407T/Q471Y 601/602T31S/C61V/A95K/V407T/ ++ ++ ++ ++ ++ A504R 603/604A95K/A286Q/S328A/V407T/ ++ ++ ++ +++ + A504R 605/606A95K/G287S/A504R/G549D ++ ++ ++ ++++ + 607/608 T31S/C61V/A95K/A286Q/ +++++ ++ + ++ S328A 609/610 C61V/S328A + ++ + ++ ++ 611/612A95K/A504R/T547Q ++ ++ ++ + + 613/614 C61V/A95K/H155S/V407T ++ ++ +++++ + ¹All activities were determined relative to reference polypeptideSEQ ID NO: 388. Levels of increased activity are defined as follows: “+”= 0.9 to 1.2; “++” > 1.2; “+++” > 2; and “++++” > 3.

EXAMPLE 8 TAL Variants of SEQ ID NO: 604

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 604, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 603, was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min and 20 μL of undiluted HTP lysate was assayed with0.1 mM tyrosine for 5 min. The results of the assays are shown in Table8-1. SEQ ID NO: 736 was selected for further directed evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 4, and 50 μL of HTP lysate diluted 2-fold was incubated with50 μL 3 g/L protease mix for 1 h at 37° C. Residual activity wasdetermined as described in Example 4. The results of this assay areshown in Table 8-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer

TAL variants were challenged with acidic buffer as described in Example5, and 40 μL of heat-treated clarified lysate was incubated with 60 μLMcIlvaine buffer pH 4. Residual activity was determined as described inExample 5. The results of this assay are shown in Table 8-1.

TABLE 8-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 604) SEQ ID Amino Acid Differences 2mM Tyr Protease 0.1 mM Tyr pH 4 NO: (nt/aa) (Relative to SEQ ID NO: 604)FIOPC FIOPC FIOPC FIOPC 615/616 N531D ++ + ++ ++ 617/618Q37A/K56G/S59A/H234Y/ ++ ++ ++ ++++ N531D 619/620 A36V/H131M + + ++ ++++621/622 Q37A/K56G/S59A/R103L/ ++ ++ ++ ++++ E145V/H234Y/A328Q/Q471Y/N531D 623/624 A36V/Q37A/H131M/E145V/ + + + ++++ H234Y/A328S 625/626A36V/Q37A/E145V/H234Y + ++ ++ ++++ 627/628 K56G/S59A/R103L/Q471Y/ + + +++++ N531D 629/630 A36V/S59A/H234Y/A328S + + ++++ 631/632Q37A/H234Y/Q471Y + ++ ++ ++++ 633/634 A36V/K56G/E145V/H234Y/ + ++ ++++++ A328S 635/636 H234Y/N531D ++ + ++ ++++ 637/638A36V/Q37A/R103L/H234Y + ++ + ++++ 639/640 H234Y/A328Q ++ ++ ++ ++++641/642 H234Y/A328S/Q471Y + ++ ++ ++++ 643/644Q37A/K56G/H234Y/N531D + + + ++++ 645/646 A36V/R103L/E145V/A328S/ + ++ ++++++ N531D 647/648 A36V/Q37A/S59A/H131M/ ++ + ++ ++++ N531D 649/650A36V/S59A/N531D + ++ ++ ++++ 651/652 K56G/S59A/Q471Y/N531D ++ ++ ++ +++653/654 Q37A/K56G/S59A/H234Y/ + ++++ A328S/R524L/N531D 655/656 H234Y +++++ 657/658 S59A/A328Q/R524L/N531D ++ + ++ ++++ 659/660A36V/H234Y/N531D + + ++ ++++ 661/662 A36V/Q37A/K56G/S59A/ + ++ + ++++E145V/H234Y/A328S 663/664 Q37A/S59A/H234Y + + + ++++ 665/666A36V/Q37A/H234Y/A328Q/ + + ++ ++++ N531D 667/668 H131M/H234Y/A328Q +++++ ++++ 669/670 A36V/Q37A/E145V/H234Y/ + + ++ ++++ A328S/Q471Y/N531D671/672 K518V/A519E/S548K/G549D ++ ++ ++ ++++ 673/674 L46T/T49D/A519E+++ ++ ++ ++ 675/676 L46R/H155L ++ ++ ++ + 677/678K33N/L46T/K518V/A534S/ ++ ++ ++ ++++ S548K/G549D 679/680 I336V/A519P ++++ ++ +++ 681/682 T49D/A519P +++ +++ ++ ++ 683/684 K33N/L46T/T49D/G549D++ ++ + ++++ 685/686 S548K/G549D + + ++ ++++ 687/688 L46T/S548K +++ ++++ +++ 689/690 G549D ++ ++ ++ ++++ 691/692 K33N/T49D/I336V +++ +++ +++++ 693/694 K33N/L46T/T49D/I336V/ ++ ++ + ++++ A519E/S548K/G549D 695/696K33N/L46T/T49D +++ +++ ++ ++ 697/698 A519P ++ + ++ ++ 699/700L46R/T49D/A519P +++ +++ ++ +++ 701/702 K33N/L46R/T49D/G549D +++ +++ ++++++ 703/704 K33N/I336V/A519P ++ ++ ++ +++ 705/706L46R/T49D/K518V/A519E/ ++ ++ ++ ++++ S548K/G549D 707/708 L46R/T49D ++ ++++ ++ 709/710 K33N/L46T/A519E ++ + ++ ++ 711/712 L541A + + 713/714 S521D++ ++ ++ ++ 715/716 Q66R/A540R ++ + ++ +++ 717/718 E523D + + + 719/720P29E/Q66N/Q111K/A540R ++ ++ ++ + 721/722 Q111K ++ ++ ++ +++ 723/724Q111K/E523A/N557E ++ ++ ++ ++ 725/726 P29E/Q111K + + + 727/728P29G/Q66N/Q111K + + + 729/730 P29E/Q111K/E523D + + + 731/732Q111K/L510K/S521D/E523A/ ++ + ++ +++ L541A/Q558D 733/734F14S/S114P/E283D/R289Q ++ ++ ++ ++++ 735/736 F14S/K56R/S114P ++ ++ ++++++ 737/738 K56R/S114P/I291V ++ ++ ++ ++++ 739/740 F14S/S114P/K518Q ++++ ++ ++++ 741/742 K56R/S114P/E206P/E283D ++ ++ ++ ++++ 743/744F14S/E283D/R289Q/I562S ++ ++ ++ + 745/746 S114P/E283D/I291V ++ ++ ++++++ 747/748 K56R/S114P/I291V/K518Q/ ++ + ++ ++++ I562S 749/750S114P/I562S ++ ++ ++ ++++ 751/752 K56R/S114P/K518Q +++ ++ ++ ++++753/754 F14S/S114P/E283S/R289Q/ ++ ++ ++ ++++ I562S 755/756S114P/E283D/R289Q/Q305E/ +++ ++ ++ ++++ K518Q 757/758 S114P/R289Q/I562S++ ++ ++ ++++ 759/760 S114P/K518Q/I562S ++ ++ ++ ++++ 761/762F14S/S114P/E283D/R289Q/ ++ ++ ++ ++++ I291V 763/764 S114P/I291V/K518Q ++++ ++ ++++ 765/766 F14S/H234L/E283D/I291V/ ++ ++ ++ ++++ Q305E 767/768K56R/K518Q ++ ++ ++ + 769/770 S114P/E283D/R289Q/I291V ++ ++ ++ ++++771/772 S114P/I291V ++ + ++ ++++ 773/774 S114P/R289Q ++ ++ ++ ++++775/776 S114P ++ ++ ++ ++++ 777/778 F14S/S114P/I291V/K518Q/ ++ + ++ ++++I562S 779/780 Q305E ++ ++ ++ + 781/782 S114P/E206P/E283S/R289Q/ ++ ++ ++++++ I562S 783/784 K56R ++ ++ ++ ++ 785/786 R289Q/I562S ++ ++ ++ ++ ¹Allactivities were determined relative to reference polypeptide SEQ ID NO:604. Levels of increased activity are defined as follows: “+” = 0.9 to1.2; “++” > 1.2; “+++” > 2; and “++++” > 3.

EXAMPLE 9 TAL Variants of SEQ ID NO: 736

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 736 for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 735, was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4 with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min and 10 μL of undiluted HTP lysate was assayed with0.1 mM tyrosine for 5 min. The results of the assays are shown in Tables9.1 and 9.2. SEQ ID NO: 790 was selected for further directed evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Protease

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 7, and residual activity was determined as described inExample 6. The results of this assay are shown in Tables 9-1 and 9-2.

HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer

TAL variants were challenged with acidic buffer as described in Example8, and residual activity was determined as described in Example 7. Theresults of this assay are shown in Tables 9-1 and 9-2.

TABLE 9-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 736)¹ SEQ ID Amino Acid Differences 2mM Tyr Protease pH 4 NO: (nt/aa) (Relative to SEQ ID NO: 736) FIOPCFIOPC FIOPC 787/788 G549D + + ++ 789/790 T49D/S59A/R289Q/I562S + ++ +791/792 S59A/E145V/H234Y/I291V + + ++ 793/794 T49D/S59A/Q66N/R289Q +++ + 795/796 R56G/H234Y/I291V + + 797/798R56G/S59A/Q111K/H234Y/N531D + + ++ 799/800 T49D/Q111K/N531D + + +801/802 Q111K/I291V + + 803/804 K518Q/N531D/G549D/I562S + + ++ 805/806R56G/S59A/E145V/R289Q/K518Q/I562S ++ ++ + 807/808 I291V/K518Q/G549D + ++809/810 R56G/S59A/H234Y + ++ ++ 811/812R56G/Q111K/E145V/H234Y/R289Q/N531D/ + + ++ I562S 813/814 K518Q + + +815/816 Q111K/E145V + ++ 817/818 T49D/R56G/Q66N/H234Y + ++ + 819/820H234Y + + ++ 821/822 T49D/R56G/Q66N/Q111K/H234Y/I291V/ + + + K518Q823/824 T49D/R56G/S59A/Q66N/I291V/N531D/ + + ++ G549D 825/826Q111K/E145V/H234Y/I291V/I562S + + ++ 827/828 Q111K/E145V/H234Y/I562S + +++ 829/830 T49D/R56G/S59A/Q111K/G549D + ++ ++ 831/832 Q111K/R289Q + + ++833/834 S59A + + ++ 835/836 R56G/Q111K/K518Q/G549D + + + 837/838T49D/Q66N/Q111K/H234Y/I291V/K518Q/ + + ++ I562S 839/840 N531D + + +841/842 R56G/S59A/Q111K/I291V/K518Q/N531D + + 843/844R56G/S59A/Q111K/E283D/K518Q/N531D + + + 845/846 T49D/H234Y + + ++847/848 S59A/H234Y/I291V + + ++ 849/850 Q111K/H234Y + + ++ 851/852D20G/S59A/H234Y/K518Q/G549D/I562S + ++ 853/854 R56G/S59A/K518Q + + +855/856 R56G/S59A/Q111K/H234Y/R289Q + ++ ++ 857/858D20G/T49D/R56G/S59A/Q111K/I291V + 859/860S59A/H234Y/I291V/I387T/G549D/I562S + + 861/862T49D/S59A/Q66N/E145V/H234Y + 863/864 Q111K/K518Q + + + 865/866H234Y/K518Q/G549D + + ++ 867/868 S59A/Q111K/ + + ++ 869/870R56G/Q111K/E145V/H234Y/K518Q/G549D/ + + ++ I562S 871/872R56G/S59A/H234Y/K518Q + + ++ 873/874 R56G/S59A/Q111K/I291V + + + ¹Allactivities were determined relative to reference polypeptide SEQ ID NO:736. Levels of increased activity are defined as follows: “+” = 0.9 to1.2; “++” > 1.2; “+++” > 2; and “++++” > 3.

TABLE 9-2 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 736)¹ SEQ ID 2 mM 0.1 mM NO: AminoAcid Differences Tyr Protease Tyr pH 4 (nt/aa) (Relative to SEQ ID NO:736) FIOPC FIOPC FIOPC FIOPC  875/876 K33N/Q37A/K518V/S521D/S548K ++ ++++ ++  877/878 Q37A/R103L/Q305E/I336V/E523D/ ++ ++ ++ +++ A540R  879/880K33N/L46R/E523D ++ ++ ++ ++  881/882 Q37A/R103L/Q305E/S521D/L541A/ ++ ++++ +++ S548K  883/884 Q37A/Q305E/I336V/A540R/S548K ++ + ++ +++  885/886L46R/R103L/Q305E/S548K ++ + ++ +++  887/888 Q37A/R103L/L541A/S548K ++ ++++ +++  889/890 Q37A/I336V/A540R ++ ++ ++ +++  891/892 Q37A/S548K ++ ++++ ++  893/894 K33N/L46R/I336V/S521D/E523D ++ ++ ++ ++  895/896K33N/Q37A/E523D/A540R/S548K ++ ++ ++ +++  897/898L46R/R103L/Q305E/Q471Y/A540R/ ++ + ++ +++ S548K  899/900 E523D ++ ++ ++ 901/902 K33N/L46R/K518V/S521D/S548K ++ ++ ++  903/904K33N/Q37A/I336V/A540R/S548K ++ ++ ++ +++  905/906 L46R/Q305E/E523D/S548K++ + ++ ++  907/908 K33N/L46R ++ ++ ++ ++  909/910 Q37A/L541A + ++ + ++ 911/912 K33N/L46R/Q305E/I336V ++ ++ ++ ++  913/914L46R/Q305E/I336V/K522E ++ + ++ ++  915/916 L46R/R103L/Q305E ++ + ++ +++ 917/918 K33N/Q37A/Q305E/A328Q/S548K ++ ++ ++ +++  919/920L46R/Q305E/Q471Y ++ + ++ ++  921/922 Q37A/Q305E/E523D/L541A/S548K ++ +++ +++  923/924 Q37A/Q305E/L541A/S548K ++ + ++ ++  925/926L46R/I336V/E523D/S548K ++ ++ ++ ++  927/928 K33N/Q37A/R103L/A519P/E523D++ ++ ++ +++  929/930 K33N/L46R/S548K ++ ++ ++ ++  931/932K33N/L46R/L541A ++ ++ ++ ++  933/934 Q37A/Q305E/Q471Y/A540R/S548K ++ +++ ++  935/936 Q37A/E523D/L541A ++ ++ ++ ++  937/938Q37A/I336V/Q471Y/L541A/S548K + ++ ++ ++  939/940K33N/L46R/R103L/Q305E/A540R ++ ++ ++ +++  941/942K33N/Q37A/R103L/K518V/E523D/ ++ ++ ++ ++ L541A  943/944K33N/Q37A/Q471Y/E523D/A540R/ ++ ++ ++ ++ S548K  945/946K33N/Q37A/R103L/Q471Y/A519P/ ++ ++ ++ +++ S548K  947/948L46R/R103L/Q305E/I336V/E523D + + ++ +++  949/950Q37A/Q471Y/S521D/A540R/S548K ++ ++ ++ ++  951/952 K33N/Q37A/S548K ++ ++++ ++  953/954 Q37A/S521D/E523D/S548K ++ ++ ++ ++  955/956K33N/L46R/I336V/S521D + ++ + ++  957/958 L46R/I336V/A540R/S548K ++ ++ ++++  959/960 Q37A/Q305E/I336V/E523D/S548K ++ + ++ +++  961/962K33N/Q37A/Q305E/I336V/S521D/ ++ ++ ++ +++ S548K  963/964Q37A/Q305E/I336V/S521D/A540R/ ++ ++ ++ +++ S548K  965/966K33N/Q37A/Q305E/A540R ++ ++ ++ +++  967/968 Q37A/Q305E/S548K ++ ++ +++++  969/970 Q37A/Q305E/Q471Y/E523D/A540R/ ++ + ++ ++ S548K  971/972K33N/Q37A/R103L/Q305E ++ ++ ++ +++  973/974 K33N/Q37A/I336V/A540R ++ ++++ +++  975/976 K33N/Q37A/K518V/S521D/A540R ++ ++ ++ ++  977/978K33N/Q37A/I336V ++ ++ ++ ++  979/980 K33N/Q37A/Q305E/A328Q ++ ++ ++ ++ 981/982 Q37A/A540R/S548K ++ ++ ++ ++  983/984 Q37A/I336V/S548K ++ ++ ++++  985/986 L46R/R103L/L541A/S548K ++ ++ ++ +++  987/988 K33N/Q37A ++ ++++ ++  989/990 Q37A/R103L/A519P/L541A/S548K ++ ++ ++ +++  991/992Q37A/R103L/A328Q/S521D/L541A/ ++ ++ ++ +++ S548K  993/994L46R/S521D/E523D ++ ++ ++ ++  995/996 K33N/Q37A/R103L/Q305E/E523D/ ++ ++++ +++ S548K  997/998 L46R/R103L/Q305E/P520Q/A540R ++ + ++ +++  999/1000L46R/Q305E/I336V/K518V ++ + ++ ++ 1001/1002 K33N/Q37A/R103L/Q305E/S548K++ + ++ +++ 1003/1004 Q37A/A519P ++ ++ ++ ++ 1005/1006 Q37A/Q471Y ++ ++++ ++ 1007/1008 L46R/Q305E ++ + ++ ++ 1009/1010 R103L ++ ++ ++ +++1011/1012 L46R/E523D/L541A/S548K ++ ++ ++ ++ 1013/1014K33N/Q37A/K518V/S548K ++ ++ ++ ++ 1015/1016 Q37A/Q471Y/K518V ++ ++ ++ ++1017/1018 K33N/L46R/R103L/A540R/S548K ++ ++ ++ +++ 1019/1020Q37A/S521D/A540R ++ ++ ++ ++ 1021/1022 K33N/L46R/R103L/Q305E/I336V/ ++ +++ +++ S548K 1023/1024 L46R/Q305E/S548K ++ ++ ++ ++ 1025/1026Q37A/Q305E/A519P/S548K ++ + ++ ++ 1027/1028 Q37A/I336V/S521D/E523D/S548K++ ++ ++ +++ 1029/1030 Q37A/E523D/S548K ++ ++ ++ ++ 1031/1032K33N/Q37A/R103L/Q305E/I336V/ ++ ++ ++ ++++ L541A/S548K 1033/1034Q37A/R103L/Q305E/Q471Y/A540R/ ++ ++ ++ +++ S548K 1035/1036K33N/Q37A/Q305E/L541A ++ ++ ++ ++ 1037/1038 Q37A/Q305E/I336V ++ ++ +++++ 1039/1040 Q37A/Q305E/S521D/A540R ++ + ++ ++ 1041/1042 S521D/S548K++ + ++ ++ 1043/1044 K33N/Q37A/R103L/A540R ++ ++ ++ +++ 1045/1046R103L/S521D/E523D/A540R + ++ + +++ 1047/1048 K33N/L46R/I336V/A540R/S548K++ ++ ++ +++ 1049/1050 Q37A/I336V ++ ++ ++ ++ 1051/1052K33N/Q37A/Q305E/Q471Y/S521D/ ++ ++ ++ ++ S548K 1053/1054 K33N/L46R/Q305E++ + ++ ++ 1055/1056 Q37A/R103L/A328Q/I336V/K518V/ ++ ++ ++ +++E523D/S548K 1057/1058 L46R/R103L/Q305E/I336V/Q471Y/ ++ ++ ++ +++A540R/S548K 1059/1060 K33N/L46R/R103L/Q305E/I336V/ ++ ++ ++ +++K518V/A519P/A540R 1061/1062 L46R/S548K ++ ++ ++ ++ 1063/1064 Q37A/R103L++ ++ + +++ 1065/1066 Q37A/R103L/Q305E/Q471Y ++ ++ ++ +++ 1067/1068K33N/L46R/I336V/A540R ++ ++ ++ +++ 1069/1070 Q37A/Q305E/L541A ++ + ++1071/1072 L46R/Q305E/I336V/S548K ++ + ++ ++ 1073/1074K33N/Q37A/S521D/S548K ++ ++ ++ ++ 1075/1076 Q37A ++ ++ ++ ++ 1077/1078K33N/Q37A/Q305E/E523D/A540R ++ ++ ++ +++ 1079/1080 L46R/Q305E/I336V ++ +++ ++ 1081/1082 K33N/Q37A/R103L/I336V/K518V/ ++ ++ ++ +++ L541A/S548K1083/1084 L46R ++ ++ ++ ++ 1085/1086 K33N/Q37A/A540R ++ ++ ++ +++1087/1088 Q37A/R103L/A540R ++ ++ ++ +++ 1089/1090 K33N/Q37A/Q305E ++ ++++ ++ 1091/1092 Q37A/R103L/I336V/S521D/L541A + ++ + +++ 1093/1094K33N/L46R/Q66K/Q305E/S521D/ + ++ +++ E523D/A540R/S548K 1095/1096K33N/Q37A/E523D ++ ++ ++ ++ 1097/1098 L46R/E523D/A540R ++ ++ ++ ++1099/1100 Q37A/R103L/Q305E/A540R ++ ++ ++ +++ 1101/1102 K33N/Q37A/R103L++ ++ ++ +++ 1103/1104 L46R/S521D/S548K ++ ++ ++ ++ 1105/1106Q37A/I336V/S521D/E523D ++ ++ ++ ++ 1107/1108 L46R/R103L/S548K ++ ++ +++++ 1109/1110 Q37A/R103L/I336V ++ ++ ++ +++ 1111/1112 L46R/L541A/S548K++ ++ + ++ 1113/1114 Q37A/Q305E ++ ++ ++ ++ 1115/1116Q37A/R103L/Q305E/I336V/S521D/ ++ + ++ +++ E523D/A540R/S548K 1117/1118K33N/L46R/Q305E/K518V/A540R/ ++ + ++ ++ S548K 1119/1120L46R/I336V/S521D/E523D ++ ++ ++ ++ 1121/1122K33N/Q37A/Q305E/I336V/S521D/ ++ ++ ++ ++ E523D/S548K 1123/1124Q305E/I336V/L541A/S548K ++ + ++ ++ 1125/1126 L46R/R103L/E523D/S548K ++++ ++ ++ 1127/1128 Q37A/Q305E/I336V/L541A/S548K ++ + ++ ++ 1129/1130Q37A/R103L/Q305E/I336V ++ + ++ +++ 1131/1132K33N/Q37A/Q305E/I336V/K518V/ ++ + ++ ++ E523D 1133/1134 L46R/R103L/L541A++ ++ + +++ 1135/1136 I336V/S548K + ++ + ++ 1137/1138R103L/I336V/A519P/S548K ++ ++ + +++ 1139/1140Q37A/Q471Y/K518V/A519P/E523D ++ ++ ++ ++ 1141/1142 A519P/S548K + + + ++1143/1144 K33N/Q37A/Q305E/I336V/Q471Y/ ++ ++ ++ ++ L541A 1145/1146K33N/Q37A/L46R/Q305E ++ + ++ ++ 1147/1148 Q37A/Q305E/S521D/E523D/A540R/++ + ++ ++ S548K 1149/1150 K33N/L46R/S521D/S548K ++ ++ ++ ++ 1151/1152Q37A/Q305E/Q471Y ++ + ++ ++ 1153/1154 Q37A/I336V/E523D/S548K + + + ++1155/1156 Q37A/Q305E/S521D/L541A/S548K ++ + ++ ++ 1157/1158K33N/R103L/I336V/K518V/S521D/ ++ ++ ++ ++ S548K 1159/1160K33N/Q37A/Q305E/I336V/L541A ++ + ++ ++ 1161/1162 K33N/Q471Y/E523D/L541A++ ++ ++ ++ 1163/1164 L46R/R103L/A540R/S548K + + + ++ 1165/1166K33N/Q37A/R103L/E523D + ++ + ++ 1167/1168 L46R/I336V/A540R ++ ++ ++ ++1169/1170 L46R/S521D/E523D/A540R ++ ++ ++ ++ 1171/1172 L46R/L541A ++ ++++ + 1173/1174 K33N/L46R/I336V/Q471Y/A519P/ ++ ++ ++ ++ E523D/S548K1175/1176 Q37A/A540R ++ ++ ++ ++ 1177/1178 L46R/T49D/N531D/G549D + + +++ 1179/1180 R289Q/S521D/E523D + + + 1181/1182H234Y/R289Q/E523D/N531D/G549D/ + + + +++ I562S 1183/1184 R289Q + + + +1185/1186 S521D + + + + 1187/1188 L46R/T49D/Q111K/H234Y/I336V + + + +++1189/1190 L46R/T49D/Q111K/H234Y/R289Q/ + + + ++ L436V/G549D 1191/1192L46R/T49D/H234Y/G549D + + + +++ 1193/1194 T49D + + + 1195/1196T49D/R289Q/Q305E/I336V/E523D + + + 1197/1198T49D/Q111K/H234Y/R289Q/I336V/ + + + ++ E523D/N531D/G549D 1199/1200S59A/G549D + + + +++ 1201/1202 T49D/S59A/H234Y/R289Q/I336V/ + + ++E523D/N531D/G549D 1203/1204 L46R/H234Y/R289Q/G549S + + + +++ 1205/1206T49D/H234Y/R289Q/E523D + + + ++ 1207/1208 S59A/Q111K/R289Q + + + +++1209/1210 R289Q/I336V/G549D + + + ++ 1211/1212 T49D/Q111K/H234Y + + ++++ 1213/1214 T49D/H234Y/Q305E/G549D + + + +++ 1215/1216H234Y/S521D/E523D/G549D + + ++ 1217/1218L46R/T49D/R289Q/E523D/N531D/ + + + ++ G549D/I562S 1219/1220T49D/Q111K/E523D/N531D/G549D ++ + + ++ 1221/1222 T49D/H234Y/G549D + ++ ++++ 1223/1224 T49D/H234Y/R289Q + ++ + ++ 1225/1226L46R/Q111K/H234Y/R289Q/N531D/ + + + +++ G549D 1227/1228Q111K/R289Q/I336V/S521D + + + ++ 1229/1230 T49D/H234Y + ++ + ++1231/1232 T49D/E523D/G549D + ++ + ++ 1233/1234L46R/T49D/Q111K/H234Y/I336V/ + ++ + +++ E523D/G549D 1235/1236T49D/Q111K/R289Q + ++ + + 1237/1238 T49D/S59A/Q111K/R289Q/E523D + ++ +++ 1239/1240 H234Y/I562S + + + ++ 1241/1242S59A/Q111K/H234Y/R289Q/Q305E/ + + + ++++ I336V/G549D 1243/1244L46R/S59A/G549D/I562S/ + ++ + +++ 1245/1246T49D/Q111K/H234Y/I336V/S521D/ + + + ++ E523D 1247/1248H234Y/R289Q/N531D + + + ++ 1249/1250 H234Y/G549D + + + +++ 1251/1252H234Y/R289Q/G549D + + + +++ 1253/1254T49D/Q111K/H234Y/R289Q/S521D/ + + + ++ E523D/N531D/G549D/I562S 1255/1256H234Y/A540R + + + +++ 1257/1258 T49D/Q111K/H234Y/I336V/E523D/ + + + ++N531D 1259/1260 H234Y/R289Q + + + ++ 1261/1262 T49D/S521D/G549D + ++ +++ 1263/1264 R289Q/S521D/E523D/G549D + + + +++ 1265/1266T49D/S59A/Q111K/H234Y/R289Q/ + ++ + +++ S521D 1267/1268T49D/R289Q/I336V/S521D/N531D + + + 1269/1270 T49D/S59A/S521D/N531D + + +++ 1271/1272 Q111K/H234Y/R289Q + + + +++ 1273/1274L46R/R289Q/G549D + + + +++ 1275/1276 T49D/S59A/H234Y/R289Q/I336V + + +++ 1277/1278 H234Y/I336V/N531D/I562S + + + +++ 1279/1280T49D/I336V/S521D/I562S + + + + 1281/1282L46R/Q111K/H234Y/S521D/G549D + + + +++ 1283/1284Q111K/S521D/E523D/G549D + + + +++ 1285/1286L46R/T49D/H234Y/I336V/S521D/ + + + +++ E523D/G549D 1287/1288T49D/S59A/I336V + + + ++ 1289/1290 S521D/I562S + + + + 1291/1292H234Y/R289Q/E523D/G549D + + + +++ 1293/1294L46R/T49D/H234Y/R289Q/I336V + + + ++ 1295/1296T49D/S521D/N531D/G549D/I562S + + + ++ 1297/1298 T49D/Q111K/H234Y/G549D +++ + +++ 1299/1300 S59A/Q111K/I336V + + + +++ 1301/1302L46R/T49D/S59A/Q111K/E523D/ + + + +++ N531D/G549D 1303/1304L46R/T49D/Q111K/I336V/G549D + + + +++ 1305/1306T49D/H234Y/R289Q/I336V/N531D/ + + + ++ G549D 1307/1308Q111K/H234Y/R289Q/E523D + + + +++ 1309/1310L46R/T49D/Q111K/H234Y/R289Q/ + + + +++ N531D/G549D 1311/1312L46R/H234Y/G549D + + + +++ 1313/1314 S59A + + + ++ 1315/1316T49D/H234Y/R289Q/E523D/N531D/ + + + ++ A540R 1317/1318T49D/S59A/R289Q/I336V + ++ + ++ 1319/1320T49D/Q111K/H234Y/R289Q/E523D/ + ++ + ++ G549D 1321/1322 L46R + + + +1323/1324 R289Q/S521D/E523D/A540R + + + + 1325/1326 G549D + + + ++1327/1328 L46R/Q111K/E523D/N531D/G549D + + + ++ 1329/1330T49D/S59A/R289Q/Q305E/I336V ++ + + ++ 1331/1332 L46R/S59A/H234Y + + ++++ 1333/1334 T49D/G549D + ++ + ++ 1335/1336 H234Y/N531D/G549D + + + +++1337/1338 T49D/Q111K/H234Y/E523D/N531D/ + + + +++ G549D 1339/1340T49D/R289Q/I336V + + + 1341/1342 S59A/Q111K/H234Y/R289Q/Q305E/ + + +++++ G549D 1343/1344 N531D/G549D + + + ++ 1345/1346Q111K/R289Q/I336V/E523D/G549D + + + +++ 1347/1348H234Y/R289Q/I336V/E523D + + + ++ 1349/1350 H234Y/I336V + + + ++1351/1352 A540R + + + + 1353/1354 T49D/Q111K/H234Y/N531D + + + ++1355/1356 H234Y + + + ++ 1357/1358 T49D/Q111K + + + + 1359/1360T49D/Q111K/N531D/G549D + + + ++ 1361/1362 L46R/G549D + + + ++ 1363/1364Q111K/I336V/I562S + ++ + ++ 1365/1366 H234Y/E523D/N531D + + + ++1367/1368 T49D/H234Y/R289Q/E523D/G549D + ++ + ++ 1369/1370T49D/Q111K/R289Q/I336V/S521D/ + ++ + ++ G549D 1371/1372T49D/H234Y/S521D + + + ++ 1373/1374 L46R/T49D/H234Y/A540R/G549D + + ++++ 1375/1376 L46R/S521D/E523D/G549D + + + ++ 1377/1378S521D/G549D + + + ++ 1379/1380 L46R/H234Y/S521D/E523D/N531D/ + + + +++A540R/G549D 1381/1382 R289Q/E523D + + + + 1383/1384H234Y/S521D/E523D + + + ++ ¹All activities were determined relative toreference polypeptide SEQ ID NO: 736. Levels of increased activity aredefined as follows: “+” = 0.9 to 1.2; “++” > 1.2; “+++” > 2; and“++++” > 3.

EXAMPLE 10 TAL Variants of SEQ ID NO: 790

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 790 for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 790 was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4 with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Tables 10-1and 10-2. SEQ ID NO: 1454 was selected for further directed evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin, whereby 50μL of HTP lysate diluted 2-fold was incubated with 50 μL 4 g/L proteasemix for 2 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker (400rpm). Protease mix consisted of 4 mg/mL chymotrypsin (Sigma Aldrich) and4 mg/mL trypsin (Sigma Aldrich) in 100 mM sodium phosphate pH 8.0.Residual activity was determined by adding 40 μL of the protease-treatedlysate to 160 μL of 200 mM sodium phosphate, 2.5 mM tyrosine, pH 7.0, ina 96-well UV-STAR® plate (Greiner Bio-One). The reactions were mixedbriefly, and activity was determined as described in Example 4. Theresults of this assay are shown in Tables 10-1 and 10-2.

TAL variants were challenged with acidic buffer, whereby 90 μL ofheat-treated clarified lysate was incubated with 90 μL McIlvaine bufferpH 4 for 1-2 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker(400 rpm). After incubation, the lysate solutions were brieflycentrifuged, and residual activity was determined by adding 20 μL of theacidic buffer-treated lysate to 180 μL of 200 mM sodium phosphate, 2.5mM tyrosine, pH 7.0, in a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and activity was determined as describedin Example 4. The results of this assay are shown in Tables 10-1 and10-2.

In some instances, the acidic buffer-treated lysate described above wasfurther challenged with protease in a combined pH and protease assay.After pretreatment with acidic buffer, the lysate solutions were brieflycentrifuged, and 75 μL of the supernatant was incubated with 75 μL 4 g/Lprotease mix dissolved in 200 mM sodium phosphate dibasic for 2 h at 37°C. in a THERMOTRON® (INFORS) titre-plate shaker (400 rpm). Residualactivity was determined by adding 40 μL of the pretreated lysate to 160μL of 200 mM sodium phosphate, 2.5 mM tyrosine, pH 7.0, in a 96-wellUV-STAR® plate (Greiner Bio-One). The reactions were mixed briefly, andactivity was determined as described in Example 4. The results of thisassay are shown in Table 10-1.

TABLE 10-1 Relative Activity of TAL Variants on Tyrosine under variousconditions (Relative to SEQ ID NO: 790)¹ SEQ ID 2 mM Combined pH NO:Amino Acid Differences (Relative Tyr Protease pH 4 and Protease (nt/aa)to SEQ ID NO: 790) FIOPC FIOPC FIOPC FIOPC 1385/1386 L202R/K332Q/D408E++ ++ ++ + 1387/1388 K44R + + + + 1389/1390 D408E ++ ++ + + 1391/1392I24T/L202R/G351N + ++ + ++ 1393/1394 I24T/K44R/R464Q + ++ + + 1395/1396I24T/K201N/L202R/G351N/Q507R + ++ + ++ 1397/1398 L202R + ++ + +1399/1400 K33N/Q37A/Q111K/S548K + ++ + + 1401/1402K33N/R56G/R103L/Q111K + ++ + + 1403/1404 L46R/I336V/A540R/G549D + ++ ++++ 1405/1406 K33N/I336V/G549D + ++ + + 1407/1408K33N/I336V/A540R/S548K + + + + 1409/1410 K33N/Q37A/L46R/R103L/Q111K/ +++ ++ ++ H234Y 1411/1412 Q37A/L46R/R56G/E523D + ++ + + 1413/1414K33N/I336V + ++ + + 1415/1416 K33N/Q37A/E523D/A540R/S548K ++ ++ + +1417/1418 K33N/Q37A/L46R/Q111K/I336V/ + + ++ ++ A540R 1419/1420K33N/L46R/R56G/H234Y/A248V/ + ++ + ++ G549D 1421/1422K33N/R103L/Q111K/H234Y + ++ + + 1423/1424 K33N/Q37A/L46R/R56G/R103L/ +++ + ++ E523D/A540R/S548K/G549D 1425/1426 K33N/Q37A/L46R/R56G/Q111K/ +++ + ++ H234Y/E523D 1427/1428 K33N/Q37A/L46R/R103L/Q111K/ + ++ + ++S548K/G549D 1429/1430 Q37A/L46R/H234Y + ++ ++ ++ 1431/1432L46R/H234Y/A540R/S548K + + + ++ 1433/1434 Q37A/Q111K/E523D + ++ + +1435/1436 K33N/L46R + ++ + ++ 1437/1438 K33N/L46R/R103L/E523D/G549D + ++++ ++ 1439/1440 K33N/H234Y/I336V/E523D/A540R/ + ++ + ++ S548K/G549D1441/1442 Q37A/R56G/H234Y/I336V/E523D/ + +++ + ++ A540R/S548K/G549D1443/1444 K33N/Q111K/H234Y/S548K + ++ + ++ 1445/1446K33N/R103L/Q111K/H234Y/I336V/ + ++ + ++ E523D/S548K 1447/1448K33N/R103L/Q111K/I336V + ++ + + 1449/1450 H234Y/E523D/A540R/S548K/ + ++++ ++ G549D 1451/1452 K33N/H234Y/E523D/S548K + ++ + ++ 1453/1454K33N/Q37A/H234Y/I336V/E523D/ + ++ + + S548K 1455/1456Q37A/L46R/I336V/E523D/A540R/ + ++ ++ ++ S548K 1457/1458 Q37A/I336V +++ + + 1459/1460 Q37A + ++ + + 1461/1462 Q37A/L46R/I336V/A540R/S548K + +++ 1463/1464 K33N/Q37A/R56G/Q111K/H234Y/ + ++ + ++ E523D/A540R/S548K1465/1466 K33N/Q37A/L46R/I336V + ++ + ++ 1467/1468 E523D/S548K + + + +1469/1470 K33N/Q37A/R56G/H234Y/E523D/ + ++ + ++ A540R/S548K 1471/1472L46R/R103L/A328Q/I336V/E523D + ++ ++ ++ 1473/1474K33N/Q37A/Q111K/E523D/A540R + ++ + + 1475/1476K33N/L46R/R56G/Q111K/S548K + ++ + ++ 1477/1478 K33N/H234Y/A540R/S548K/ +++ ++ ++ G549D 1479/1480 Q37A/S548K/G549D + ++ + ++ 1481/1482K33N/H234Y/E523D/A540R/ + ++ ++ ++ S548K 1483/1484K33N/Q37A/L46R/R56G/Q111K/ + ++ + ++ E523D/A540R 1485/1486K33N/Q37A/L46R/S548K/G549D + ++ ++ ++ 1487/1488K33N/Q37A/L46R/Q111K/H234Y/ + ++ ++ ++ A328Q/E523D/A540R/S548K 1489/1490I336V/E523D + ++ + + 1491/1492 K33N/Q37A/R103L/Q111K/H234Y/ + ++ + ++I336V/A540R/S548K 1493/1494 L46R/H234Y/I336V ++ ++ ++ ++ 1495/1496K33N/R103L/Q111K/H234Y/A540R/ + ++ + ++ G549D 1497/1498K33N/Q37A/Q111K/H234Y/E523D/ + ++ + ++ A540R/S548K 1499/1500Q37A/R103L/I336V/S548K/G549D + ++ + ++ 1501/1502Q37A/H234Y/A328Q/I336V/E523D + ++ + + 1503/1504K33N/H234Y/A328Q/E523D/A540R/ + ++ + ++ G549D 1505/1506K33N/Q37A/L46R/H234Y/E523D + ++ ++ ++ 1507/1508K33N/I336V/E523D/A540R/S548K/ + ++ ++ ++ G549D 1509/1510L46R/R103L/I336V + ++ ++ ++ 1511/1512 K33N/Q37A/L46R/R103L/E523D + ++ ++++ 1513/1514 K33N/Q37A/R103L/I336V/E523D/ + ++ + ++ A540R/S548K1515/1516 Q37A/R56G/I336V/E523D/A540R/ + ++ + ++ S548K ¹All activitieswere determined relative to the reference polypeptide of SEQ ID NO: 790.Levels of increased activity are defined as follows: ““+”” = 0.9 to 1.2;“++” > 1.2; and “+++” > 2

TABLE 10-2 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 790)¹ SEQ ID NO: Amino AcidDifferences 2 mM tyr Protease pH 4 (nt/aa) (Relative to SEQ ID NO: 790)FIOPC FIOPC FIOPC 1517/1518 S424A + 1519/1520 M102P +++ + 1521/1522G417A ++ ++ + 1523/1524 L418V ++ + + 1525/1526 I420L + + + 1527/1528S402A + ++ + 1529/1530 T391N + ++ + 1531/1532 M392A + + + 1533/1534L313M ++ + + 1535/1536 L210T ++ ++ + 1537/1538 D530V ++ ++ + 1539/1540L313I ++ ++ ++ 1541/1542 I74A ++ ++ + 1543/1544 L415F ++ + 1545/1546R506C + + + ¹All activities were determined relative to the referencepolypeptide of SEQ ID NO: 790. Levels of increased activity are definedas follows: ““+”” = 0.9 to 1.2; “++” > 1.2; and “+++” > 2.

EXAMPLE 11 TAL Variants of SEQ ID NO: 1454

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 1454 for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 1454 was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4 with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 11-1,and SEQ ID NO: 1578 was selected for further directed evolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 10. Residual activity was determined as described in Example10, and the results of this assay are shown in Table 11-1.

TAL variants were challenged with acidic buffer, whereby 50 μL ofheat-treated clarified lysate was incubated with 50 μL McIlvaine bufferpH 3.8 for 1.5 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker(400 rpm). After incubation, the lysate solutions were brieflycentrifuged, and residual activity was determined by adding 40 μL of theacidic buffer-treated lysate to 160 μL of 200 mM sodium phosphate, 2.5mM tyrosine, pH 7.0, in a 96-well UV-STAR® plate (Greiner Bio-One). Thereactions were mixed briefly, and activity was determined as describedin Example 10. The results of this assay are shown in Table 11-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay. After 30 μL ofheat-treated clarified lysate was incubated with 30 μL McIlvaine bufferpH 3.8 for 1.5 h at 37° C., 60 μL 4 g/L protease mix dissolved in 200 mMsodium phosphate dibasic was added, and the resulting solution wasincubated for 2 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker(400 rpm). Residual activity was determined as described in Example 10,and the results of this assay are shown in Table 11-1.

TABLE 11-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 1454)¹ Amino Acid Combined SEQDifferences 2 mM pH and ID NO: (Relative to Tyr Protease pH 3.8 Protease(nt/aa) SEQ ID NO: 1454) FIOPC FIOPC FIOPC FIOPC 1547/1548I24T/K44R/G549D ++ ++ + 1549/1550 I24T/L46R/Q111K/ + ++ ++ + R506C1551/1552 I24T/K44R/L46R/ ++ ++ + L202R/T391N 1553/1554 Q111K/L202R/ +++ +++ + L313M/G417A/ A540R/G549D 1555/1556 K44R/R103L/ ++ ++ +Q111K/L313M/ T391N/A540R 1557/1558 L313M/T391N/ ++ + A540R/G549D1559/1560 I24T/K44R/L202R/ ++ ++ + L313M/T391N/ A540R 1561/1562I24T/L46R/R103L/ ++ ++ + T391N/R506C 1563/1564 K44R/L46R/R103L/ ++ ++ +Q111K/T391N 1565/1566 Q111K/L202R/ ++ ++ + T391N 1567/1568I24T/K44R/Q111K/ + + ++ + L202R/R464Q/ R506C 1569/1570 L202R/L313M/ ++++ + T391N/G549D 1571/1572 L46R/Q111K/R506C + ++ ++ + 1573/1574L202R/R506C/ + +++ + A540R/G549D 1575/1576 R103L/L313M/ + + + ++ G549D1577/1578 I24T/L46R/M102P/ + ++ ++ ++ L313M/R506C 1579/1580 I24T/L202R +++ ++ + 1581/1582 Q111K/L202R/ + ++ ++ + L313M/T391N/ R506C 1583/1584I24T/L46R/R103L/ + ++ +++ ++ Q111K/R464Q/ R506C/G549D 1585/1586 G417A +++ + + 1587/1588 L202R/R506C/ + + +++ + A540R 1589/1590 Q111K + ++ + ++1591/1592 I24T + ++ + + 1593/1594 I24T/L46R/Q111K/ + ++ + L313M/T391N1595/1596 Q111K/L202R/ + ++ ++ + L313M/T391N/ R464Q 1597/1598L46R/R103L/R506C + + +++ + 1599/1600 L202R/G417A/ + ++ +++ ++R464Q/A540R/ G549D 1601/1602 K44R/L46R/R103L/ + ++ ++ ++ L202R/L313M/T391N 1603/1604 I24T/R103L/L202R + ++ +++ ++ 1605/1606 I24T/L46R/L202R/++ + + T391N/R506C 1607/1608 I24T/L46R/G549D + ++ +++ ++ 1609/1610K44R/L46R/R103L/ + + ++ + L202R/G417A/ R464Q 1611/1612 L46R/L313M/ + ++++ + T391N/A540R 1613/1614 L46R/L202R/ + +++ + R506C/G549D 1615/1616Q111K/L202R/ ++ ++ + T391N/A540R 1617/1618 K44R/Q111K/ + + ++ +R464Q/R506C 1619/1620 Q111K/L202R ++ ++ +++ ++ 1621/1622 I24T/K44R + +++ + 1623/1624 L202R/R464Q/ + ++ ++ + R506C 1625/1626 L46R/Q111K/ + ++++ + G417A 1627/1628 L46R/L202R + ++ +++ ++ 1629/1630I24T/L46R/Q111K/ + + +++ ++ L313M/G549D 1631/1632 Q111K/R464Q/ + ++ + ++A540R/G549D 1633/1634 K44R/L46R/M102P/ + ++ ++ ++ L202R/L313M/ G417A¹All activities were determined relative to the reference polypeptideSEQ ID NO: 1454. Levels of increased activity are defined as follows:““+”” = 0.9 to 1.2; “++” > 1.2; and “+++” > 2

EXAMPLE 12 TAL Variants of SEQ ID NO: 1578

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 1578, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 1578 was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 8-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 12-1.Based on the results, SEQ ID NO: 1660 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 10. The residual activity was determined as described inExample 10. The results of this assay are shown in Table 12-1.

TAL variants were challenged with acidic buffer, whereby 70 μL ofheat-treated clarified lysate was incubated with 70 μL McIlvaine bufferpH 4 for 1.5 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker(400 rpm). The residual activity was determined as described in Example10. The results of this assay are shown in Table 12-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay. Specifically, lysatechallenged with acidic buffer as described above was centrifuged, and 50μL of the supernatant was incubated with 50 μL 4 g/L protease mixdissolved in 100 mM sodium phosphate pH 8. The resulting solution wasincubated for 2 h at 37° C. in a THERMOTRON® (INFORS) titre-plate shaker(400 rpm), and the residual activity was determined as described inExample 10. The results of this assay are shown in Table 12-1.

TABLE 12-1 Relative Activity of TAL Variants on Tyrosine under VariousConditions (Relative to SEQ ID NO: 1578)¹ Amino Acid Combined SEQ IDDifferences 2 mM pH and NO: (Relative to Tyr Protease pH 4 Protease(nt/aa) SEQ ID NO: 1578) FIOPC FIOPC FIOPC FIOPC 1635/1636D20G/K44R/L202R/ ++ ++ ++ ++ C506R 1637/1638 R103L/L202R/ ++ ++ ++ ++A540R 1639/1640 Q111K ++ ++ ++ ++ 1641/1642 R103L/R464Q/ + ++ + ++ G549D1643/1644 Q111K/C506R ++ ++ ++ ++ 1645/1646 K44R/L202R/ ++ ++ ++ ++R464Q/C506R 1647/1648 R464Q/C506R ++ ++ ++ ++ 1649/1650 Q111K/L202R/ ++++ ++ ++ M313L/Q471Y/ C506R 1651/1652 C506R ++ ++ ++ ++ 1653/1654R103L/Q471Y ++ ++ + ++ 1655/1656 M313L/R464Q/ + ++ + ++ Q471Y/C506R1657/1658 L202R/R464Q/ ++ ++ ++ ++ C506R 1659/1660 L202R/R464Q/ ++ ++ ++++ C506R/G549D 1661/1662 R103L/Q111K/ ++ ++ + ++ Q471Y 1663/1664R56G/Q111K/ ++ ++ ++ ++ C506R 1665/1666 C506R/G549D + ++ ++ ++ 1667/1668R103L/Q111K/ ++ ++ ++ ++ L202R/R464Q/ Q471Y 1669/1670 D20G/L202R/ ++ ++++ ++ M313L/Q471Y/ C506R 1671/1672 L202R ++ ++ ++ ++ 1673/1674Q111K/L202R/ ++ ++ ++ ++ Q471Y 1675/1676 R56G/C506R ++ ++ ++ ++1677/1678 K44R/Q111K/ ++ ++ ++ ++ L202R/R464Q/ Q471Y/C506R/ G549D1679/1680 D20G/R103L/ ++ ++ ++ ++ Q111K/Q471Y/ C506R 1681/1682L202R/M313L/ ++ ++ ++ ++ R464Q/Q471Y/ C506R 1683/1684 Q111K/L202R/ ++++ + + M313L/L316Q 1685/1686 T24S ++ ++ ++ ++ 1687/1688 R504A ++ ++ ++++ 1689/1690 Y41L ++ ++ ++ ++ 1691/1692 R464S ++ ++ ++ ++ 1693/1694A540R ++ ++ ++ ++ 1695/1696 Y41N ++ ++ ++ ++ 1697/1698 R464N ++ ++ ++ ++1699/1700 R504L ++ ++ ++ ++ 1701/1702 Y41H ++ ++ + ++ 1703/1704 Q66K ++++ ++ ++ 1705/1706 E51H ++ ++ ++ ++ 1707/1708 Q305E ++ ++ ++ ++1709/1710 R46S ++ ++ ++ ++ 1711/1712 A59S ++ ++ ++ ++ 1713/1714 K44E ++++ ++ ++ 1715/1716 Q66T ++ ++ ++ ++ 1717/1718 T24K ++ ++ ++ ++ 1719/1720Q305D ++ ++ ++ ++ 1721/1722 Y41S ++ ++ + ++ 1723/1724 K44R ++ ++ ++ ++1725/1726 Y41Q ++ ++ + ++ 1727/1728 T48A ++ ++ + + 1729/1730 D523H ++ ++++ ++ 1731/1732 Q66N ++ ++ ++ ++ 1733/1734 A36S ++ ++ ++ + 1735/1736A59N ++ ++ ++ + 1737/1738 K50M ++ ++ ++ ++ 1739/1740 K44S ++ ++ ++ +1741/1742 A519P ++ ++ ++ ++ 1743/1744 A519E ++ ++ ++ + 1745/1746 D27S ++++ ++ + 1747/1748 A36T ++ ++ ++ + 1749/1750 A519K ++ ++ ++ ++ 1751/1752K44P ++ ++ ++ ++ 1753/1754 T24E ++ ++ ++ ++ 1755/1756 K518S ++ ++ ++ ++1757/1758 C500L ++ ++ ++ ++ 1759/1760 T48G ++ ++ + + 1761/1762 A540H ++++ ++ ++ 1763/1764 L208M ++ ++ ++ ++ 1765/1766 Q66H/W528R ++ ++ ++ ++1767/1768 T547G ++ ++ ++ ++ 1769/1770 A519T ++ ++ ++ + 1771/1772 Y41A ++++ + ++ 1773/1774 T24Q ++ ++ + ++ 1775/1776 R46A ++ ++ + + 1777/1778Q55S ++ ++ ++ ++ 1779/1780 E51A ++ ++ ++ ++ 1781/1782 Pl 97 V ++ ++ + ++1783/1784 N33E ++ ++ ++ ++ 1785/1786 T547M/V553I ++ ++ ++ ++ 1787/1788P272A ++ ++ ++ ++ 1789/1790 A331G ++ ++ ++ ++ 1791/1792 A543T ++ ++ ++++ 1793/1794 A543V ++ ++ ++ ++ 1795/1796 I556L ++ ++ ++ ++ 1797/1798A555S ++ ++ ++ ++ 1799/1800 Q470I ++ ++ ++ ++ 1801/1802 V559Y ++ ++ ++++ 1803/1804 Q289L ++ ++ ++ ++ 1805/1806 K332N ++ ++ ++ ++ 1807/1808R292L/V553S + ++ + + 1809/1810 Q470T ++ ++ ++ ++ 1811/1812 Y432L ++ ++++ ++ 1813/1814 S435T ++ ++ ++ ++ 1815/1816 C506E ++ ++ ++ ++ 1817/1818V559C ++ ++ ++ ++ 1819/1820 I556V ++ ++ ++ ++ 1821/1822 V559I ++ ++ ++++ 1823/1824 V559H ++ ++ ++ ++ 1825/1826 V559M ++ ++ ++ ++ 1827/1828Y432T ++ ++ ++ ++ 1829/1830 V559L ++ ++ ++ ++ 1831/1832 S312C/V559Y ++++ ++ ++ 1833/1834 S435A ++ ++ ++ ++ ¹All activities were determinedrelative to the reference polypeptide of SEQ ID NO: 1578. Levels ofincreased activity are defined as follows: ““+”” = 0.9 to 1.2; and“++” > 1.2.

EXAMPLE 13 TAL Variants of SEQ ID NO: 1660

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 1660, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 1660 was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 8-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 13-1.Based on the results, SEQ ID NO: 1844 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin, whereby 50μL of HTP lysate diluted 2 or 2.5-fold was incubated with 50 μL 4 g/Lprotease mix for 2 h at 37° C., in a THERMOTRON® (INFORS) titre-plateshaker (400 rpm). Protease mix consisted of 4 mg/mL chymotrypsin (SigmaAldrich) and 4 mg/mL trypsin (Sigma Aldrich) in 100 mM sodium phosphatepH 8.0. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 13-1.

TAL variants were challenged with acidic buffer as described in Example12. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 13-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay, as described inExample 12. Residual activity was determined as described in Example 10.The results of this assay are shown in Table 13-1.

TABLE 13-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 1660)¹ Amino Acid Combined SEQ IDDifferences 2 mM pH and NO: (Relative to Tyr Protease pH 4 Protease(nt/aa) SEQ ID NO: 1660) FIOPC FIOPC FIOPC FIOPC 1835/1836 A36S/P197V/++ ++ ++ ++ Q305D/K332N/ C500L 1837/1838 Q305D/K332N/ ++ ++ ++ ++G457S/C500L 1839/1840 A36S/Q55S/Q305D/ +++ ++ ++ ++ K332N/P410M1841/1842 A36S/P197V/ ++ ++ ++ ++ Q305D/K332N/ P410M 1843/1844A36S/Q305D/ +++ ++ ++ ++ K332N/P410M 1845/1846 A36S/Q305D/ ++ ++ ++ ++P410M 1847/1848 A36S/P197V/ ++ ++ ++ ++ Q305D/K332N/ P410M/V559I1849/1850 Q305D/K332N/ +++ ++ ++ ++ P410M 1851/1852 Q305D ++ ++ ++ ++1853/1854 A36S/Q66N/P197V/ ++ ++ ++ ++ Q305D/K332N/ G457S 1855/1856Q55S/Q305D/ ++ ++ ++ ++ K332N/G457S/ A543V/T547G 1857/1858 Q66N/Q305D/++ ++ ++ ++ K332N 1859/1860 A36S/P197V/ ++ ++ ++ ++ Q305D/P410M1861/1862 Q305D/C500L/ ++ ++ ++ ++ A543V/T547G 1863/1864 Q305D/K332N/+++ ++ +++ ++ P410M/C500L 1865/1866 P197V/Q305D ++ ++ ++ ++ 1867/1868A36S/Q66N/P197V/ +++ ++ ++ ++ Q305D/P410M 1869/1870 A36S/P197V/Q305D ++++ ++ ++ 1871/1872 A36S/Q66N/Q305D/ +++ ++ +++ ++ K332N/P410M 1873/1874P197V/Q305D/ +++ ++ +++ ++ P410M 1875/1876 Q305D/K332N ++ ++ ++ ++1877/1878 A36S/P197V/ ++ +++ ++ ++ Q305D/C500L 1879/1880 A36S/Q305D/ ++++ ++ ++ K332N 1881/1882 A36S/Q66N/Q305D/ ++ ++ ++ ++ C500L 1883/1884Q55S/Q305D/ ++ ++ ++ ++ C500L/V559I 1885/1886 Q305D/K332N/ ++ ++ ++ ++C500L 1887/1888 P197V/Q305D/ +++ ++ ++ ++ K332N/P410M 1889/1890Q305D/P410M +++ ++ +++ ++ 1891/1892 A36S/P197V/ +++ ++ +++ ++Q305D/K332N/ P410M/C500L 1893/1894 Q55S/Q305D ++ ++ ++ ++ 1895/1896A36S/Q305D ++ ++ ++ ++ 1897/1898 P197V/Q305D/ ++ ++ ++ ++ K332N/C500L/A543V/T547G 1899/1900 Q66N/Q305D ++ ++ ++ ++ 1901/1902 A36S/Q66N/Q305D++ ++ ++ ++ 1903/1904 P197V/Q305D/ ++ ++ ++ ++ C500L 1905/1906S134C/R202L/ ++ ++ ++ ++ Q305D 1907/1908 Q305D/K332N/ ++ ++ ++ +G457S/A555S 1909/1910 Q305D/K332N/ ++ ++ ++ ++ G457S 1911/1912P272A/S435T/ ++ ++ ++ ++ Y526L 1913/1914 L208M/P272A/ ++ ++ ++ + Y432L1915/1916 R46S/Q111K/ ++ ++ ++ ++ L208M/P272A/ S435T/Y526L 1917/1918Q111K/P272A/ ++ ++ ++ ++ Q471Y/Y526L 1919/1920 L208M ++ ++ ++ ++1921/1922 Q111K/P272A/ ++ ++ ++ ++ Y432T/S435T 1923/1924 Y432L/Y526L ++++ ++ ++ 1925/1926 D20G/P197V/ ++ ++ ++ ++ Y432T/A519K 1927/1928K44E/R46S/Q111K/ ++ ++ ++ ++ L208M 1929/1930 Q111K/Y526L ++ + ++ +1931/1932 Q111K + + + + 1933/1934 S435T + + + + 1935/1936 Q471Y + + + +1937/1938 R46S/Y432L/Q471Y + + + + 1939/1940 K44E/R46S/Q111K/ + + + +Y432T/A519K 1941/1942 Q111K/P272A/ + + + + A519K 1943/1944 L208M/P410M/++ + ++ ++ S435T 1945/1946 Y41L/Y432T/Y526L ++ ++ ++ + 1947/1948T24E/R46S/P410M/ ++ ++ ++ ++ S435T/K518S 1949/1950 D20G ++ ++ ++ ++1951/1952 L208M/S435T/ ++ ++ ++ ++ A519K 1953/1954 S435T/A519K/ ++ ++ ++++ Y526L 1955/1956 R46S/L208M/ ++ ++ ++ ++ P272A/Y432L/ S435T/Y526L1957/1958 P272A ++ ++ + ++ 1959/1960 K44E/Q111K/ ++ ++ ++ ++Y432L/A519K/ Y526L 1961/1962 D20G/K44E/L208M/ + ++ + + P272A/A519K1963/1964 T24E + ++ + + 1965/1966 D20G/Q111K/ ++ ++ ++ ++ L208M/P272A/P410M/Y432T 1967/1968 Q111K/Y432T/ ++ ++ ++ +++ Y526L 1969/1970Q471Y/Y526L ++ ++ ++ ++ 1971/1972 A519K ++ ++ ++ ++ 1973/1974K44E/L208M/ ++ ++ ++ ++ Y432T/S435T 1975/1976 S435T/K518S ++ ++ ++ ++1977/1978 Q111K/Y432L/ ++ ++ + ++ S435T 1979/1980 Y41L/K44E/R46S/ ++ ++++ + Q111K/P272A 1981/1982 L211Q/P410I + + + + 1983/1984 S298G/P410I ++++ ++ + 1985/1986 P410L + + + + ¹All activities were determined relativeto the reference polypeptide of SEQ ID NO: 1660. Levels of increasedactivity are defined as follows: ““+”” = 0.9 to 1.2; “++” > 1.2; and“+++” > 2.

EXAMPLE 14 TAL Variants of SEQ ID NO: 1844

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 1844, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 1844 was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 14-1.Based on the results, SEQ ID NO: 2030 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin, whereby 50μL of HTP lysate diluted 2.5-fold was incubated with 50 μL 4 g/Lprotease mix for 2 h at 37° C. in a THERMOTRON® (INFORS) titre-plateshaker (400 rpm). Protease mix consisted of 4 mg/mL chymotrypsin (SigmaAldrich) and 4 mg/mL trypsin (Sigma Aldrich) in 100 mM sodium phosphatepH 8.0. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 14-1.

TAL variants were challenged with acidic buffer as described in Example12. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 14-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay. Specifically, lysatechallenged with acidic buffer as described above was centrifuged, and 40μL of the supernatant was incubated with 60 μL 4 g/L protease mixdissolved in 100 mM sodium phosphate pH 8. The resulting solution wasincubated for 2 h at 37° C., in a THERMOTRON® (INFORS) titre-plateshaker (400 rpm), and residual activity was determined as described inExample 10. The results of this assay are shown in Table 14-1.

TABLE 14-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 1844)¹ Amino Acid Combined SEQ IDDifferences 2 mM pH and NO: (Relative to SEQ ID Tyr Protease pH 4Protease (nt/aa) NO: 1844) FIOPC FIOPC FIOPC FIOPC 1987/1988 Q111K/Q289L+++ +++ ++ +++ 1989/1990 Q289L/R504A +++ +++ ++ +++ 1991/1992A59S/Q289L/S435T +++ +++ ++ +++ 1993/1994 Q111K/Q289L/ +++ +++ ++ +++C500L 1995/1996 Q111K/Y432L +++ +++ ++ +++ 1997/1998 Q289L/S435T/ ++++++ ++ +++ R504A/I556L 1999/2000 A59S/Q289L +++ +++ ++ +++ 2001/2002Q111K +++ +++ ++ ++ 2003/2004 Q289L +++ +++ ++ +++ 2005/2006Q111K/Y432L/ ++ ++ ++ ++ C500L 2007/2008 Q289L/S435T/ +++ +++ ++ +++C500L/R504A 2009/2010 A59S/Y432L/S435T/ ++ ++ ++ ++ R504A/I556L2011/2012 A59S/Q66T/Q289L/ ++ ++ ++ ++ C500L/R504A/I556L 2013/2014S435T/R504A ++ ++ ++ ++ 2015/2016 Q289L/Y432L ++ ++ ++ ++ 2017/2018Q111K/Q289L/ ++ ++ ++ ++ S435T/C500L 2019/2020 A59S/Q66T/Q111K/ ++ ++ ++++ Q289L/Y432L/ C500L 2021/2022 A59S/Q111K/ +++ ++ ++ ++ Y432L/C500L2023/2024 T24K/A59S/Q111K/ ++ ++ ++ ++ Q289L/R504A 2025/2026 Q66T/Q111K+++ ++ ++ ++ 2027/2028 T24K/Q111K/Q289L ++ ++ ++ ++ 2029/2030Q289L/Y432L/ ++ +++ ++ ++ S435T 2031/2032 T24K/Q289L ++ ++ ++ ++2033/2034 T24K/Q111K/ ++ ++ ++ ++ Q289L/C500L/ R504A 2035/2036T24K/Q111K/ +++ ++ ++ ++ S435T/C500L 2037/2038 R504A/I556L ++ ++ ++ ++2039/2040 Q111K/A331G/ ++ ++ ++ +++ Y432L/R504A 2041/2042 Q111K/C500L/+++ ++ ++ ++ R504A 2043/2044 Q111K/A331G/ ++ ++ ++ ++ S435T/C500L2045/2046 A59S/Q66T/Q111K/ +++ ++ ++ ++ Y432L/C500L 2047/2048A59S/Q66T/Q111K/ +++ +++ ++ ++ Y432L/R504A 2049/2050 T24K/Q111K/Y432L ++++ ++ ++ 2051/2052 A59S/Y432L ++ +++ ++ +++ 2053/2054 Q111K/Q289L/ +++++ ++ ++ S435T 2055/2056 R504A ++ +++ ++ ++ 2057/2058 Q289L/S435T +++++ ++ ++ 2059/2060 T24K/Y432L/S435T/ ++ ++ ++ ++ R504A 2061/2062A59S/Q111K ++ +++ ++ ++ 2063/2064 Y432L ++ ++ ++ ++ 2065/2066L211Q/K518S/ +++ ++ ++ ++ A519T/T547G 2067/2068 Y41Q/L208M/ +++ ++ ++ ++T547G 2069/2070 L208M/L211Q/ ++ ++ ++ ++ T547G 2071/2072Y41Q/E51A/M410L/ ++ ++ ++ ++ A543V 2073/2074 E51A/A519T ++ ++ ++ ++2075/2076 L208M ++ ++ ++ ++ 2077/2078 L211Q/T547G ++ ++ ++ ++ 2079/2080L211Q ++ ++ ++ ++ 2081/2082 Y41Q/L208M/ ++ ++ ++ ++ K518S/A519T/ T547G2083/2084 Y41Q/M410L/ ++ ++ + ++ K518S/T547G 2085/2086 T547G ++ ++ ++ ++2087/2088 L208M/L211Q/ ++ ++ ++ ++ M410L/A519T 2089/2090Y41Q/K44P/L208M/ ++ ++ ++ ++ T547G 2091/2092 K44R/L208M/ ++ ++ ++ ++K518S/T547G 2093/2094 Y41Q/L208M/ ++ ++ ++ ++ M410L/A543V/ T547G2095/2096 Y41Q/M410L/ ++ ++ + ++ K518S/A543V 2097/2098 A519T ++ ++ ++ ++2099/2100 M410L ++ ++ + ++ 2101/2102 Y41Q/A543V +++ ++ ++ ++ ¹Allactivities were determined relative to the reference polypeptide of SEQID NO: 1844. Levels of increased activity are defined as follows: ““+””= 0.9 to 1.2; “++” > 1.2; and “+++” > 2.

EXAMPLE 15 TAL Variants of SEQ ID NO: 2030

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 2030, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 2030, was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 15-1.Based on the results, SEQ ID NO: 2114 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 14. Residual activity was determined as described in Example10. The results of this assay are shown in Table 15-1.

TAL variants were challenged with acidic buffer as described in Example12, with the exception that lysate was incubated with McIlvaine buffer,pH 3.8. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 15-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay. Specifically, lysatechallenged with acidic buffer as described above was centrifuged, and 50μL of the supernatant was incubated with 50 μL 4 g/L pancreatindissolved in 100 mM sodium phosphate pH 8. The resulting solution wasincubated for 2 h at 37° C., in a THERMOTRON® (INFORS) titre-plateshaker (400 rpm), and residual activity was determined as described inExample 10. The results of this assay are shown in Table 15-1.

TABLE 15-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 2030)¹ Amino Acid Combined SEQ IDDifferences 2mM pH and NO: (Relative to SEQ Tyr Protease pH 3.8 Protease(nt/aa) ID NO: 2030) FIOPC FIOPC FIOPC FIOPC 2103/2104 A45V/L244M/ +++ + + A324V/K513R 2105/2106 A45V/L436I/ + ++ + + I512V/K513R 2107/2108K13Q/A45V/ + ++ + + I512V 2109/2110 K13S/V22T/V/ + ++ + + I512V A452111/2112 K13S/A45V + ++ + + 2113/2114 A45 V/1512V + ++ + + 2115/2116Q8D ++ ++ + + 2117/2118 L428V ++ ++ + + 2119/2120 TUG + ++ + + 2121/2122A4D ++ ++ + + 2123/2124 A45V + ++ + + 2125/2126 T17Q + ++ + + 2127/2128L251V + ++ + + 2129/2130 T11D ++ ++ + ++ 2131/2132 K13R + ++ + +2133/2134 T11A + ++ + + 2135/2136 T11S + ++ + + 2137/2138 Q8R ++ ++ + +2139/2140 T11K + + + + 2141/2142 V341A + + + + 2143/2144 S19H + + + +2145/2146 A324R + + + + 2147/2148 Q8G + + + + 2149/2150 I144L + +2151/2152 I327M + + + + ¹All activities were determined relative to thereference polypeptide of SEQ ID NO: 2030. Levels of increased activityare defined as follows: ““+”” = 0.9 to 1.2; and “++” > 1.2.

EXAMPLE 16 TAL Variants of SEQ ID NO: 2114

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 2114, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 2114, was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4, with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 16-1.Based on the results, SEQ ID NO: 2156 was selected for further directedevolution.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 15. Residual activity was determined as described in Example10. The results of this assay are shown in Table 16-1.

TAL variants were challenged with acidic buffer as described in Example15. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 16-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay as described inExample 15, and residual activity was determined as described in Example10. The results of this assay are shown in Table 16-1.

TABLE 16-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 2114)¹ Amino Acid DifferencesCombined SEQ (Relative to 2 mM pH and ID NO: SEQ ID NO: Tyr Protease pH3.8 Protease (nt/aa) 2114) FIOPC FIOPC FIOPC FIOPC 2153/2154 Q8D/T11D/++ ++ ++ ++ Q111K/R504A 2155/2156 Q8D/T11D/ ++ ++ ++ ++ T17Q/V22T/A324V/R504A/ A519T 2157/2158 A4D ++ ++ ++ ++ 2159/2160 A4D/L428V ++ ++++ ++ 2161/2162 A4D/Q8R/T11D/ ++ ++ ++ ++ K13R/V22T/ Q111K/R504A2163/2164 A4D/A324V ++ ++ ++ ++ 2165/2166 T11D/T17Q/ ++ ++ ++ ++V22T/A324V 2167/2168 A4D/Q8D/T11G/ ++ ++ ++ ++ T17P/A324V/ L428V/R504A2169/2170 A4D/Q8D/V22T/ ++ ++ ++ ++ Q111K/R504A 2171/2172 A4D/T17Q/ ++++ + ++ A241S/A519T/ T547A 2173/2174 T11G/K13R/ ++ ++ ++ ++ R504A2175/2176 Q8D/T11D/ ++ ++ ++ ++ K13R/A324V 2177/2178 A4D/Q8R/T11D/ ++ ++++ ++ V22T/Q111K 2179/2180 A236V ++ ++ + + 2181/2182 I228M ++ ++ + +2183/2184 H16Q ++ ++ + ++ 2185/2186 T437G ++ ++ + ++ 2187/2188 F10G ++++ ++ ++ 2189/2190 L316T + ++ 2191/2192 I2T ++ ++ ++ ++ 2193/2194I228M/Q259H ++ ++ + + 2195/2196 D305E/I307N/ ++ ++ + + I425V 2197/2198V385A ++ ++ + ++ 2199/2200 T7Q ++ ++ + + 2201/2202 I2G ++ ++ + +2203/2204 T430E + ++ + + 2205/2206 M478L + ++ + + 2207/2208 Q6G ++++ + + 2209/2210 V57I ++ ++ + + 2211/2212 A39S ++ ++ + + 2213/2214 1425V++ ++ + + 2215/2216 H16T ++ + + + 2217/2218 T505S + + + + 2219/2220 S14G++ + + + ¹All activities were determined relative to the referencepolypeptide of SEQ ID NO: 2114. Levels of increased activity are definedas follows: ““+”” = 0.9 to 1.2; “++” > 1.2; and “+++” > 2.

EXAMPLE 17 TAL Variants of SEQ ID NO: 2156

In this Example, experiments for evolution and screening of TAL variantsderived from SEQ ID NO: 2156, for improved tyrosine activity, proteaseresistance, and low pH tolerance are described. Directed evolution ofthe TAL encoded by SEQ ID NO: 2156, was carried out by constructinglibraries of variant genes. These libraries were then plated, grown, andscreened using the methods described below.

HTP Activity Analysis of Clarified Lysates

HTP growth and lysis of E. coli cells expressing TAL variants wereperformed as described in Example 4. TAL variant activity on tyrosinewas assayed as described in Example 4 with the following conditions: 20μL of HTP lysate diluted 10-fold in water was assayed with 2.2 mMtyrosine for 5 min. The results of the assays are shown in Table 17-1.

HTP Activity Analysis of Clarified Lysates Pretreated with Proteaseand/or Acidic Buffer

TAL variants were challenged with chymotrypsin and trypsin as describedin Example 15. Residual activity was determined as described in Example10. The results of this assay are shown in Table 17-1.

TAL variants were challenged with acidic buffer as described in Example15. Residual activity was determined as described in Example 10. Theresults of this assay are shown in Table 17-1.

In some instances, acidic buffer-treated lysate was further challengedwith protease in a combined pH and protease assay as described inExample 15, and residual activity was determined as described in Example10. The results of this assay are shown in Table 17-1.

TABLE 17-1 Relative Activity of TAL Variants on Tyrosine Under VariousConditions (Relative to SEQ ID NO: 2156)¹ Amino Acid Combined SEQDifferences 2 mM pH and ID NO: (Relative to SEQ Tyr Protease pH 3.8Protease (nt/aa) ID NO: 2156) FIOPC FIOPC FIOPC FIOPC 2221/2222Q111K/M478L + + + 2223/2224 A241S/V385A/ + + M478L 2225/2226H16Q/V57I + + + 2227/2228 V57I/Q111K/P114S + + + + 2229/2230 T437G + + +2231/2232 H16Q/V385A/ + + M478L/T547G 2233/2234 A241S/V385A/ + +M478L/T547G 2235/2236 H16Q/V385A/ + ++ + + T437G 2237/2238Q111K/A241S/T + + + + 437G/M478L 2239/2240 A241S/V385A + ++ + +2241/2242 D20G/V385A/ + ++ + T437G/M478L/ T547G 2243/2244 V57I + + + +2245/2246 V385A + ++ + + 2247/2248 V385A/T437G/ + + + M478L/T547G2249/2250 H16Q/A241S/T437G + ++ + + 2251/2252 H16Q ++ ++ + + 2253/2254A241S/T437G + ++ + + 2255/2256 V57I/T437G + + + + 2257/2258 H16Q/V385A +++ + + 2259/2260 T437G/M478L + + + + 2261/2262 A4D/H16Q/V385A/ + ++ + +M478L 2263/2264 A4D/V57I/Q111K/ ++ ++ + + T437G 2265/2266Q111K/P114S/ + + + + R202L/Y234H/ L289Q/D305Q/ M313L/V324A/ N332K/V336I/M410P/L432Y/ T435S/Q464R/ A504R/V512I/ T519A/D523E/ K548S/D549G2267/2268 H16Q/Y41Q/T437G ++ + + + 2269/2270 F10G/H16Q/T437G + + + +2271/2272 A4D/T7Q/V57I/ ++ ++ + ++ Q111K 2273/2274T7Q/V385A/T547G + + + + 2275/2276 H16Q/Y41Q/V57I/ + + + T437G/M478L2277/2278 M478L + + + + 2279/2280 A241S ++ + + + 2281/2282H16Q/Y41Q/V57I/ + + + Q111K/V385A/ T547G 2283/2284 H16Q/T437G + + + +2285/2286 A4D/T7Q/Q111K/ + + + + P114S/T437G/ T547G 2287/2288V57I/Q111K/ + + + + M478L/T547G 2289/2290 V385A/T437G + + + + ¹Allactivities were determined relative to the reference polypeptide of SEQID NO: 2156. Levels of increased activity are defined as follows: ““+””= 0.9 to 1.2; and “++” > 1.2.

While the invention has been described with reference to the specificembodiments, various changes can be made and equivalents can besubstituted to adapt to a particular situation, material, composition ofmatter, process, process step or steps, thereby achieving benefits ofthe invention without departing from the scope of what is claimed.

For all purposes in the United States of America, each and everypublication and patent document cited in this disclosure is incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute an admission as to its contents or date.

What is claimed is:
 1. A recombinant tyrosine ammonia lyase and/orbiologically active recombinant tyrosine ammonia lyase fragmentcomprising an amino acid sequence comprising at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to SEQ ID NO:
 2. 2. Therecombinant tyrosine ammonia lyase of claim 1, wherein said tyrosineammonia lyase comprises at least one substitution at position or set ofpositions selected from 79/95/107, 79/107, 79/107/410, 79/410,95/107/184, 95/107/184/534, 95/184/534, 99/107/184, 107/184/534, and107/401/534, wherein the positions are numbered with reference to SEQ IDNO:
 2. 3. The recombinant tyrosine ammonia lyase of claim 1, whereinsaid tyrosine ammonia lyase comprises at least one substitution in atleast one position or set of positions selected from 28, 29, 33,33/34/37/46/279/554, 33/34/37/90/554, 33/34/46/66/90/289,33/34/90/289/554, 33/34/279/289, 33/66, 33/66/289, 33/66/289/554,34/37/46/289, 34/37/66/279/289, 34/46, 34/56/90, 34/56/90/289,34/66/289, 34/90/289/554, 34/90/519, 36/70/234/302/461/500, 36/79,36/79/304/407, 36/79/407/461/500/531, 36/111/304/461/531,36/407/461/500/531, 37, 46, 46/66/289/519/554, 49, 56, 66,66/90/279/289/554, 66/90/289/554, 69, 70/79/234/361/407,70/111/461/500/531, 70/304/407/461, 72, 79/111/234/361/500,79/111/234/531, 79/111/361, 79/111/531, 79/304/361, 79/304/461/531,79/407/461, 88, 90/289/519, 90/289/554, 131, 142, 198, 201, 272, 278,279/289/554, 289/519/554, 289/554, 295, 304/361, 305, 350, 361/500/531,461/500, 490, 491, 499, 510, 518, 519, 521, 522, 523, 524, 531, 540,541, 544, 547, 548, 554, 557, 558, and 560, wherein the positions arenumbered with reference to SEQ ID NO:
 14. 4. The recombinant tyrosineammonia lyase of claim 1, wherein said tyrosine ammonia lyase comprisesat least one substitution in at least one position or set of positionsselected from 28/34/49/70/234/289/544/554, 33/34/49/234/531/547/554,33/34/66/70/519/531, 33/34/66/289/522/531, 33/34/70/234/289/519,33/34/70/234/518/519/547/554, 33/34/70/289/522/554, 33/34/289/522/531,34/49/66/531, 34/49/234/531/554, 34/49/289/544/547,34/66/70/234/518/519/547, 34/66/531/554, 34/66/554, 34/70/234/289/522,34/70/234/554, 34/70/289/522, 34/234/522/554, 34/289, 34/289/522/531,34/289/522/544/554, 34/522, 34/544, 66/70/234, and 234/531/544/547,wherein the positions are numbered with reference to SEQ ID NO:
 86. 5.The recombinant tyrosine ammonia lyase of claim 1, wherein said tyrosineammonia lyase comprises at least one substitution in at least oneposition or set of positions selected from 31, 37/49/56/66/111,37/49/56/234/557, 37/49/56/500/524, 37/49/66/111/234, 37/49/111/524,37/111, 37/111/361/500/557/558, 37/234, 43/59/93/506, 43/306,43/306/506, 49/56/66/111/153/234/500/524, 49/66, 56/111/234, 56/234/500,61, 65, 65/336, 66, 66/111/234/500, 93/506, 95, 103, 103/107/417/421,103/306/506, 103/421, 104/105, 107, 107/417/421, 111/234/548/558,111/361, 145, 146, 155, 172, 197, 211, 234, 234/524, 237/238/506, 286,304, 306, 306/506, 328, 336, 361/524/557/558, 407, 417/421, 421, 471,504, 506, 549, and 558, wherein the positions are numbered withreference to SEQ ID NO:
 334. 6. The recombinant tyrosine ammonia lyaseof claim 1, wherein said tyrosine ammonia lyase comprises at least onesubstitution in at least one position or set of positions selected from31/61/95/286/328, 31/61/95/407/504, 31/61/95/471/504, 31/61/407/504,31/95/155/286/504/549, 31/95/237/504/547/549, 31/95/286/504,31/95/286/504/549, 31/95/328/504, 31/95/504, 31/95/549, 37/49/56/59/145,37/103/111/145/421/500/524, 49/56/59/234/500, 49/59/66/111/145/234,49/59/145/234/421, 56/59/66/234, 56/59/111/145, 56/59/111/145/234/421,56/66/145/500, 56/103/111/145/234/500, 59/103/145/234/421/500,59/103/234/421, 59/145, 59/234, 61, 61/95/155/286/407/471,61/95/155/407, 61/95/286, 61/95/286/328/504, 61/95/328/407,61/95/328/549, 61/95/407, 61/95/504/547, 61/286/407, 61/328, 61/328/504,61/328/504/549, 61/407, 61/504, 66/103/145/234/421,95/155/286/328/407/504, 95/155/286/549, 95/286, 95/286/328/407/504,95/286/328/504, 95/286/547, 95/287/504/549, 95/328/504, 95/407,95/407/504, 95/504/547, 95/549, 103/111/145/234/421, 111/145/234,111/234, 145/234/500, 286/328/407/504/549, and 549, wherein thepositions are numbered with reference to SEQ ID NO:
 388. 7. Therecombinant tyrosine ammonia lyase of claim 1, wherein said tyrosineammonia lyase comprises at least one substitution in at least oneposition or set of positions selected from 14/56/114, 14/114/283/289,14/114/283/289/291, 14/114/283/289/562, 14/114/291/518/562, 14/114/518,14/234/283/291/305, 14/283/289/562, 29/66/111, 29/66/111/540, 29/111,29/111/523, 33/46/49, 33/46/49/336/519/548/549, 33/46/49/549,33/46/518/534/548/549, 33/46/519, 33/49/336, 33/336/519,36/37/56/59/145/234/328, 36/37/59/131/531, 36/37/103/234,36/37/131/145/234/328, 36/37/145/234, 36/37/145/234/328/471/531,36/37/234/328/531, 36/56/145/234/328, 36/59/234/328, 36/59/531,36/103/145/328/531, 36/131, 36/234/531,37/56/59/103/145/234/328/471/531, 37/56/59/234/328/524/531,37/56/59/234/531, 37/56/234/531, 37/59/234, 37/234/471, 46/49,46/49/518/519/548/549, 46/49/519, 46/155, 46/548, 49/519, 56,56/59/103/471/531, 56/59/471/531, 56/114/206/283, 56/114/291,56/114/291/518/562, 56/114/518, 56/518, 59/328/524/531, 66/540, 111,111/510/521/523/541/558, 111/523/557, 114, 114/206/283/289/562,114/283/289/291, 114/283/289/305/518, 114/283/291, 114/289, 114/289/562,114/291, 114/291/518, 114/518/562, 114/562, 131/234/328, 234, 234/328,234/328/471, 234/531, 289/562, 305, 336/519, 518/519/548/549, 519, 521,523, 531, 541, 548/549, and 549, wherein the positions are numbered withreference to SEQ ID NO:
 604. 8. The recombinant tyrosine ammonia lyaseof claim 1, wherein said tyrosine ammonia lyase comprises at least onesubstitution in at least one position or set of positions selected from20/49/56/59/111/291, 20/59/234/518/549/562, 49/56/59/66/291/531/549,49/56/59/111/549, 49/56/66/111/234/291/518, 49/56/66/234,49/59/66/145/234, 49/59/66/289, 49/59/289/562,49/66/111/234/291/518/562, 49/111/531, 49/234, 56/59/111/234/289,56/59/111/234/531, 56/59/111/283/518/531, 56/59/111/291,56/59/111/291/518/531, 56/59/145/289/518/562, 56/59/234, 56/59/234/518,56/59/518, 56/111/145/234/289/531/562, 56/111/145/234/518/549/562,56/111/518/549, 56/234/291, 59, 59/111, 59/145/234/291, 59/234/291,59/234/291/387/549/562, 111/145, 111/145/234/291/562, 111/145/234/562,111/234, 111/289, 111/291, 111/518, 234, 234/518/549, 291/518/549, 518,518/531/549/562, 531, and 549, wherein the positions are numbered withreference to SEQ ID NO:
 736. 9. The recombinant tyrosine ammonia lyaseof claim 1, wherein said tyrosine ammonia lyase comprises at least onesubstitution in at least one position or set of positions selected from33/37, 33/37/46/305, 33/37/103, 33/37/103/305,33/37/103/305/336/541/548, 33/37/103/305/523/548, 33/37/103/305/548,33/37/103/336/518/541/548, 33/37/103/471/519/548, 33/37/103/518/523/541,33/37/103/519/523, 33/37/103/523, 33/37/103/540, 33/37/305,33/37/305/328, 33/37/305/328/548, 33/37/305/336/471/541,33/37/305/336/518/523, 33/37/305/336/521/523/548, 33/37/305/336/521/548,33/37/305/336/541, 33/37/305/471/521/548, 33/37/305/523/540,33/37/305/540, 33/37/305/541, 33/37/336, 33/37/336/540,33/37/336/540/548, 33/37/471/523/540/548, 33/37/518/521/540,33/37/518/521/548, 33/37/518/548, 33/37/521/548, 33/37/523,33/37/523/540/548, 33/37/540, 33/37/548, 33/46,33/46/66/305/521/523/540/548, 33/46/103/305/336/518/519/540,33/46/103/305/336/548, 33/46/103/305/540, 33/46/103/540/548, 33/46/305,33/46/305/336, 33/46/305/518/540/548, 33/46/336/471/519/523/548,33/46/336/521, 33/46/336/521/523, 33/46/336/540, 33/46/336/540/548,33/46/518/521/548, 33/46/521/548, 33/46/523, 33/46/541, 33/46/548,33/103/336/518/521/548, 33/471/523/541, 37, 37/103, 37/103/305/336,37/103/305/336/521/523/540/548, 37/103/305/336/523/540, 37/103/305/471,37/103/305/471/540/548, 37/103/305/521/541/548, 37/103/305/540,37/103/328/336/518/523/548, 37/103/328/521/541/548, 37/103/336,37/103/336/521/541, 37/103/519/541/548, 37/103/540, 37/103/541/548,37/305, 37/305/336, 37/305/336/521/540/548, 37/305/336/523/548,37/305/336/540/548, 37/305/336/541/548, 37/305/471,37/305/471/523/540/548, 37/305/471/540/548, 37/305/519/548,37/305/521/523/540/548, 37/305/521/540, 37/305/521/541/548,37/305/523/541/548, 37/305/541, 37/305/541/548, 37/305/548, 37/336,37/336/471/541/548, 37/336/521/523, 37/336/521/523/548, 37/336/523/548,37/336/540, 37/336/548, 37/471, 37/471/518, 37/471/518/519/523,37/471/521/540/548, 37/519, 37/521/523/548, 37/521/540, 37/523/541,37/523/548, 37/540, 37/540/548, 37/541, 37/548, 46,46/49/59/111/523/531/549, 46/49/111/234/289/436/549,46/49/111/234/289/531/549, 46/49/111/234/336, 46/49/111/234/336/523/549,46/49/111/336/549, 46/49/234/289/336, 46/49/234/336/521/523/549,46/49/234/540/549, 46/49/234/549, 46/49/289/523/531/549/562,46/49/531/549, 46/59/234, 46/59/549/562, 46/103/305,46/103/305/336/471/540/548, 46/103/305/336/523, 46/103/305/471/540/548,46/103/305/520/540, 46/103/305/548, 46/103/523/548, 46/103/540/548,46/103/541, 46/103/541/548, 46/103/548, 46/111/234/289/531/549,46/111/234/521/549, 46/111/523/531/549, 46/234/289/549,46/234/521/523/531/540/549, 46/234/549, 46/289/549, 46/305, 46/305/336,46/305/336/518, 46/305/336/522, 46/305/336/548, 46/305/471,46/305/523/548, 46/305/548, 46/336/521/523, 46/336/523/548, 46/336/540,46/336/540/548, 46/521/523, 46/521/523/540, 46/521/523/549, 46/521/548,46/523/540, 46/523/541/548, 46/541, 46/541/548, 46/548, 46/549, 49,49/59/111/234/289/521, 49/59/111/289/523, 49/59/234/289/336,49/59/234/289/336/523/531/549, 49/59/289/305/336, 49/59/289/336,49/59/336, 49/59/521/531, 49/111, 49/111/234,49/111/234/289/336/523/531/549, 49/111/234/289/521/523/531/549/562,49/111/234/289/523/549, 49/111/234/336/521/523, 49/111/234/336/523/531,49/111/234/523/531/549, 49/111/234/531, 49/111/234/549, 49/111/289,49/111/289/336/521/549, 49/111/523/531/549, 49/111/531/549, 49/234,49/234/289, 49/234/289/336/531/549, 49/234/289/523,49/234/289/523/531/540, 49/234/289/523/549, 49/234/305/549, 49/234/521,49/234/549, 49/289/305/336/523, 49/289/336, 49/289/336/521/531,49/336/521/562, 49/521/531/549/562, 49/521/549, 49/523/549, 49/549, 59,59/111/234/289/305/336/549, 59/111/234/289/305/549, 59/111/289,59/111/336, 59/549, 103, 103/336/519/548, 103/521/523/540, 111/234/289,111/234/289/523, 111/289/336/521, 111/289/336/523/549, 111/336/562,111/521/523/549, 234, 234/289, 234/289/336/523, 234/289/523/531/549/562,234/289/523/549, 234/289/531, 234/289/549, 234/336, 234/336/531/562,234/521/523, 234/521/523/549, 234/523/531, 234/531/549, 234/540,234/549, 234/562, 289, 289/336/549, 289/521/523, 289/521/523/540,289/521/523/549, 289/523, 305/336/541/548, 336/548, 519/548, 521,521/548, 521/549, 521/562, 523, 531/549, 540, and 549, wherein thepositions are numbered with reference to SEQ ID NO:
 736. 10. Therecombinant tyrosine ammonia lyase of claim 1, wherein said tyrosineammonia lyase comprises at least one substitution in at least oneposition or set of positions selected from 24/44/464,24/201/202/351/507, 24/202/351, 33/37/46/56/103/523/540/548/549,33/37/46/56/111/234/523, 33/37/46/56/111/523/540, 33/37/46/103/111/234,33/37/46/103/111/548/549, 33/37/46/103/523,33/37/46/111/234/328/523/540/548, 33/37/46/111/336/540,33/37/46/234/523, 33/37/46/336, 33/37/46/548/549,33/37/56/111/234/523/540/548, 33/37/56/234/523/540/548,33/37/103/111/234/336/540/548, 33/37/103/336/523/540/548,33/37/111/234/523/540/548, 33/37/111/523/540, 33/37/111/548,33/37/234/336/523/548, 33/37/523/540/548, 33/46, 33/46/56/111/548,33/46/56/234/248/549, 33/46/103/523/549, 33/56/103/111, 33/103/111/234,33/103/111/234/336/523/548, 33/103/111/234/540/549, 33/103/111/336,33/111/234/548, 33/234/328/523/540/549, 33/234/336/523/540/548/549,33/234/523/540/548, 33/234/523/548, 33/234/540/548/549, 33/336,33/336/523/540/548/549, 33/336/540/548, 33/336/549, 37, 37/46/56/523,37/46/234, 37/46/336/523/540/548, 37/46/336/540/548,37/56/234/336/523/540/548/549, 37/56/336/523/540/548,37/103/336/548/549, 37/111/523, 37/234/328/336/523, 37/336, 37/548/549,44, 46/103/328/336/523, 46/103/336, 46/234/336, 46/234/540/548,46/336/540/549, 202, 202/332/408, 234/523/540/548/549, 336/523, 408, and523/548, wherein the positions are numbered with reference to SEQ ID NO:790.
 11. The recombinant tyrosine ammonia lyase of claim 1, wherein saidtyrosine ammonia lyase comprises at least one substitution in at leastone position or set of positions selected from 74, 102, 210, 313, 391,392, 402, 415, 417, 418, 420, 424, 506, and 530, wherein the positionsare numbered with reference to SEQ ID NO:
 790. 12. The recombinanttyrosine ammonia lyase of claim 1, wherein said tyrosine ammonia lyasecomprises at least one substitution in at least one position or set ofpositions selected from 24, 24/44, 24/44/46/202/391,24/44/111/202/464/506, 24/44/202/313/391/540, 24/44/549,24/46/102/313/506, 24/46/103/111/464/506/549, 24/46/103/391/506,24/46/111/313/391, 24/46/111/313/549, 24/46/111/506, 24/46/202/391/506,24/46/549, 24/103/202, 24/202, 44/46/102/202/313/417, 44/46/103/111/391,44/46/103/202/313/391, 44/46/103/202/417/464, 44/103/111/313/391/540,44/111/464/506, 46/103/506, 46/111/417, 46/111/506, 46/202,46/202/506/549, 46/313/391/540, 103/313/549, 111, 111/202,111/202/313/391/464, 111/202/313/391/506, 111/202/313/417/540/549,111/202/391, 111/202/391/540, 111/464/540/549, 202/313/391/549,202/417/464/540/549, 202/464/506, 202/506/540, 202/506/540/549,313/391/540/549, and 417, wherein the positions are numbered withreference to SEQ ID NO:
 1454. 13. The recombinant tyrosine ammonia lyaseof claim 1, wherein said tyrosine ammonia lyase comprises at least onesubstitution in at least one position or set of positions selected from20/44/202/506, 20/103/111/471/506, 20/202/313/471/506, 24, 27, 33, 36,41, 44, 44/111/202/464/471/506/549, 44/202/464/506, 46, 48, 50, 51, 55,56/111/506, 56/506, 59, 66, 66/528, 103/111/202/464/471, 103/111/471,103/202/540, 103/464/549, 103/471, 111, 111/202/313/316,111/202/313/471/506, 111/202/471, 111/506, 197, 202,202/313/464/471/506, 202/464/506, 202/464/506/549, 208, 272, 289,292/553, 305, 312/559, 313/464/471/506, 331, 332, 432, 435, 464,464/506, 470, 500, 504, 506, 506/549, 518, 519, 523, 540, 543, 547,547/553, 555, 556, and 559, wherein the positions are numbered withreference to SEQ ID NO:
 1578. 14. The recombinant tyrosine ammonia lyaseof claim 1, wherein said tyrosine ammonia lyase comprises at least onesubstitution in at least one position or set of positions selected from20, 20/44/208/272/519, 20/111/208/272/410/432, 20/197/432/519, 24,24/46/410/435/518, 36/55/305/332/410, 36/66/197/305/332/457,36/66/197/305/410, 36/66/305, 36/66/305/332/410, 36/66/305/500,36/197/305, 36/197/305/332/410, 36/197/305/332/410/500,36/197/305/332/410/559, 36/197/305/332/500, 36/197/305/410,36/197/305/500, 36/305, 36/305/332, 36/305/332/410, 36/305/410,41/44/46/111/272, 41/432/526, 44/46/111/208, 44/46/111/432/519,44/111/432/519/526, 44/208/432/435, 46/111/208/272/435/526,46/208/272/432/435/526, 46/432/471, 55/305, 55/305/332/457/543/547,55/305/500/559, 66/305, 66/305/332, 111, 111/272/432/435,111/272/471/526, 111/272/519, 111/432/435, 111/432/526, 111/526,134/202/305, 197/305, 197/305/332/410, 197/305/332/500/543/547,197/305/410, 197/305/500, 208, 208/272/432, 208/410/435, 208/435/519,211/410, 272, 272/435/526, 298/410, 305, 305/332, 305/332/410,305/332/410/500, 305/332/457, 305/332/457/500, 305/332/457/555,305/332/500, 305/410, 305/500/543/547, 410, 432/526, 435, 435/518,435/519/526, 471, 471/526, and 519, wherein the positions are numberedwith reference to SEQ ID NO:
 1660. 15. The recombinant tyrosine ammonialyase of claim 1, wherein said tyrosine ammonia lyase comprises at leastone substitution in at least one position or set of positions selectedfrom 24/59/111/289/504, 24/111/289, 24/111/289/500/504, 24/111/432,24/111/435/500, 24/289, 24/432/435/504, 41/44/208/547, 41/51/410/543,41/208/410/543/547, 41/208/518/519/547, 41/208/547, 41/410/518/543,41/410/518/547, 41/543, 44/208/518/547, 51/519, 59/66/111/289/432/500,59/66/111/432/500, 59/66/111/432/504, 59/66/289/500/504/556, 59/111,59/111/432/500, 59/289, 59/289/435, 59/432, 59/432/435/504/556, 66/111,111, 111/289, 111/289/435, 111/289/435/500, 111/289/500,111/331/432/504, 111/331/435/500, 111/432, 111/432/500, 111/500/504,208, 208/211/410/519, 208/211/547, 211, 211/518/519/547, 211/547, 289,289/432, 289/432/435, 289/435, 289/435/500/504, 289/435/504/556,289/504, 410, 432, 435/504, 504, 504/556, 519, and 547, wherein thepositions are numbered with reference to SEQ ID NO:
 1844. 16. Therecombinant tyrosine ammonia lyase of claim 1, wherein said tyrosineammonia lyase comprises at least one substitution in at least oneposition or set of positions selected from 4, 8, 11, 13, 13/22/45/512,13/45, 13/45/512, 17, 19, 45, 45/244/324/513, 45/436/512/513, 45/512,144, 251, 324, 327, 341, and 428, wherein the positions are numberedwith reference to SEQ ID NO:
 2030. 17. The recombinant tyrosine ammonialyase of claim 1, wherein said tyrosine ammonia lyase comprises at leastone substitution in at least one position or set of positions selectedfrom 4/7/57/111, 4/7/111/114/437/547, 4/16/385/478, 4/57/111/437,7/385/547, 10/16/437, 16, 16/41/57/111/385/547, 16/41/57/437/478,16/41/437, 16/57, 16/241/437, 16/385, 16/385/437, 16/385/478/547,16/437, 20/385/437/478/547, 57, 57/111/114, 57/111/478/547, 57/437,111/114/202/234/289/305/313/324/332/336/410/432/435/464/504/512/519/523/548/549,111/241/437/478, 111/478, 241, 241/385, 241/385/478, 241/385/478/547,241/437, 385, 385/437, 385/437/478/547, 437, 437/478, and 478, whereinthe positions are numbered with reference to SEQ ID NO:
 2156. 18. Arecombinant tyrosine ammonia lyase comprising a sequence that is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the polypeptide sequence of SEQ ID NO: 2, 14, 86, 334, 388,604, 736, 790, 1454, 1578, 1660, 1844, 2030, and/or
 2156. 19. Therecombinant tyrosine ammonia lyase of claim 1, wherein said recombinanttyrosine ammonia lyase is thermostable.
 20. The recombinant tyrosineammonia lyase of claim 1, wherein said recombinant tyrosine ammonialyase is resistant to proteolysis.
 21. The recombinant tyrosine ammonialyase of claim 1, wherein said recombinant tyrosine ammonia lyase isacid stable.
 22. The recombinant tyrosine ammonia lyase of claim 1,wherein said recombinant tyrosine ammonia lyase is purified.
 23. Therecombinant tyrosine ammonia lyase of claim 1, wherein said recombinanttyrosine ammonia lyase exhibits at least one improved property selectedfrom: i) enhanced catalytic activity; ii) reduced sensitivity toproteolysis; iii) increased tolerance to acidic pH; iv) reducedaggregation; v) decreased Km for tyrosine; vi) decreased immunogenicity;or a combination of any of i), ii), iii), iv), v), and/or vi), ascompared to a reference sequence selected from SEQ ID NO: 2, 14, 86,334, 388, 604, 736, 790, 1454, 1578, 1660, 1844, 2030, 2114, and/or2156.
 24. A composition comprising at least one recombinant tyrosineammonia lyase of claim
 1. 25. A recombinant polynucleotide sequenceencoding at least one recombinant tyrosine ammonia lyase set forth inclaim
 1. 26. The recombinant polynucleotide sequence of claim 25,wherein said polynucleotide sequence is codon-optimized.
 27. Anexpression vector comprising a recombinant polynucleotide sequenceencoding at least one recombinant tyrosine ammonia lyase of claim
 1. 28.The expression vector of claim 27, wherein said recombinantpolynucleotide sequence is operably linked to a control sequence.
 29. Ahost cell comprising the expression vector of claim
 27. 30. The hostcell of claim 29, wherein said host cell is selected from prokaryoticand eukaryotic cells.
 31. A method of producing a recombinant tyrosineammonia lyase, comprising culturing said host cell of claim 29, underconditions that said recombinant tyrosine ammonia lyase encoded by saidrecombinant polynucleotide is produced.
 32. The method of claim 31,further comprising the steps of recovering and purifying saidrecombinant tyrosine ammonia lyase.
 33. A pharmaceutical composition forthe treatment of tyrosinemia, comprising the composition of claim 24.34. The pharmaceutical composition of claim 33, further comprising apharmaceutically acceptable carrier and/or excipient.
 35. Thepharmaceutical composition of claim 33, wherein said composition issuitable for oral administration to a human.
 36. The pharmaceuticalcomposition of claim 33, wherein said composition is suitable forparenteral injection into a human
 37. A method for treating and/orpreventing the symptoms of tyrosinemia or alkaptonuria in a subject,comprising providing a subject having tyrosinemia or alkaptonuria, andproviding the pharmaceutical composition of claim 33, to said subject.38. A method for the production of L-tyrosine and/or L-tyrosinederivatives comprising the steps of providing at least one recombinanttyrosine ammonia lyase of claim 1, and a suitable substrate, andcombining said recombinant tyrosine ammonia lyase and said substrateunder conditions such that L-tyrosine and/or at least one L-tyrosinederivative is produced.
 39. A method for the production of coumaricacid, comprising the steps of providing at least one recombinanttyrosine ammonia lyase of claim 1, and a suitable substrate, andcombining said recombinant tyrosine ammonia lyase and said substrateunder conditions such that coumaric acid is produced.